I. INTRODUCTION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTION <<II. RESULTSIII. DISCUSSIONIV. CONCLUSIONSV. MATERIALS AND METHODSSUPPLEMENTARY MATERIALThe field of wearable electronics is growing rapidly and is guided by an awareness of users' safety and the environmental impact of electronic products due to their numbers and short life cycle. The materials and manufacturing processes are the principle concerns when adopting green electronics concepts for novel technology development. This is further accentuated in the case of electronic wearables that interface with humans, where, for hygiene reasons, devices are required to be both disposable and biocompatible. With the aim of achieving a sustainable, green approach to designing wearable devices, multiple considerations need to be taken into account at the design stage of the electronics.11. C. Santato, M. I. Vladu, M. Holuszko, and L. Yin, Adv. Sustainable Syst. 6, 2100333 (2022). https://doi.org/10.1002/adsu.202100333 For an efficient use of resources, the design and manufacturing of devices need to include an effective recycling pathway, make use of biocompatible and biosourced materials, and result in minimal waste production.Inkjet printing is a highly efficient, non-contact, additive, solution-based patterning technique with low-cost, high-quality, and high-throughput advantages.2,32. K. Yan, J. Li, L. Pan, and Y. Shi, APL Mater. 8, 120705 (2020). https://doi.org/10.1063/5.00316693. M. Caironi and Y.-Y. Noh, In Large Area and Flexible Electronics ( John Wiley & Sons, 2015), p. 588. Owing to these characteristics, it became the reference method for the manufacturing of functional materials, especially those that are chemically incompatible with other microfabrication processes on flexible and wearable substrates.44. A. Hussain, N. Abbas, and A. Ali, Chemosensors 10, 103 (2022). https://doi.org/10.3390/chemosensors10030103 Inkjet printing stands out for its high-resolution (10–100 μm) digital patterning while avoiding precious material waste.55. Y. Bonnassieux, C. J. Brabec, Y. Cao, T. B. Carmichael, M. L. Chabinyc, K.-T. Cheng, G. Cho, A. Chung, C. L. Cobb, A. Distler, H.-J. Egelhaaf, G. Grau, X. Guo, G. Haghiashtiani, T.-C. Huang, M. M. Hussain, B. Iniguez, T.-M. Lee, L. Li, Y. Ma, D. Ma, M. C. McAlpine, T. N. Ng, R. Österbacka, S. N. Patel, J. Peng, H. Peng, J. Rivnay, L. Shao, D. Steingart, R. A. Street, V. Subramanian, L. Torsi, and Y. Wu, Flexible Printed Electron. 6, 023001 (2021). https://doi.org/10.1088/2058-8585/abf986 As it is a drop-on-demand deposition technique only a small amount (in the picoliter range66. Fujifilm Dimatix Materials Printer DMP-2800 Series User Manual, 2.0. Fujifilm, USA.) of active materials are dispensed to create the design. This process has allowed for the printing of various functional materials and the fabrication of novel wearable devices. Examples range from wearable displays to health monitoring sensors7,87. M. Pietsch, S. Schlisske, M. Held, N. Strobel, A. Wieczorek, and G. Hernandez-Sosa, J. Mater. Chem. C 8, 16716 (2020). https://doi.org/10.1039/D0TC04627B8. M. Galliani, L. M. Ferrari, and E. Ismailova, J. Vis. Exp. (185), e63204 (2022). https://doi.org/10.3791/63204 and even green electronic memory cells.99. I. Salaoru, S. Maswoud, and S. Paul, Micromachines 10, 417 (2019). https://doi.org/10.3390/mi10060417Inkjet printing uses bespoke solution-based materials, specifically formulated to match the process' governing multiplex physics.10,1110. A. Teichler, J. Perelaer, and U. S. Schubert, J. Mater. Chem. C 1, 1910 (2013). https://doi.org/10.1039/c2tc00255h11. A. Soleimani-Gorgani, Adv. Nat. Sci. 9, 025009 (2018). https://doi.org/10.1088/2043-6254/aac2a0 The optimum balance of the inks' physical and chemical properties yields stable fluid jetting, reduced nozzle clogging, and a uniform dispensed film morphology.12–1412. H. Hu and R. G. Larson, J. Phys. Chem. B 110, 7090 (2006). https://doi.org/10.1021/jp060923213. D. Lohse, Annu. Rev. Fluid Mech. 54, 349 (2022). https://doi.org/10.1146/annurev-fluid-022321-11400114. D. Lohse and X. Zhang, Nat. Rev. Phys. 2, 426 (2020). https://doi.org/10.1038/s42254-020-0199-z Among the wide variety of organic and inorganic printable materials available, conducting polymers are frequently chosen for flexible and bio-electronic devices fabrication. In the last decade, conducting polymers (CPs) have been extensively employed due to their mechanical and chemical properties, enabling their fast, low-cost processability.1515. Z. Rahimzadeh, S. M. Naghib, Y. Zare, and K. Y. Rhee, J. Mater. Sci. 55, 7575 (2020). https://doi.org/10.1007/s10853-020-04561-2 Among CPs, poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) is one of the most used p-type semiconductors, both in industry and academics, owing to its high conductivity, chemical stability, semi-transparency, and, most importantly, commercial availability.16,1716. C. Boehler, Z. Aqrawe, and M. Asplund, Bioelectron. Med. 2, 89 (2019). https://doi.org/10.2217/bem-2019-001417. M. N. Gueye, A. Carella, J. Faure-Vincent, R. Demadrille, and J.-P. Simonato, Prog. Mater. Sci. 108, 100616 (2020). https://doi.org/10.1016/j.pmatsci.2019.100616 The commercial PEDOT:PSS is a water dispersion with a solid content of active material typically not exceeding 5%.1818. W. Lövenich, Polym. Sci., Ser. C 56, 135 (2014). https://doi.org/10.1134/S1811238214010068 The aqueous mixture is blended with additives to fine-tune its chemical, physical, and electrical properties toward targeted applications, while at the same time accommodating the specifics of a particular patterning method. The reported formulations based on commercial PEDOT:PSS have been typically proposed to achieve high-end electrical properties in photovoltaics applications.1919. M. Chen, A. Iyer, and R. Opila, in IEEE 46th Photovoltaic Specialists Conference (PVSC), 2019. Studies describing the role of different additives in the PEDOT:PSS inks, supported by surface tension measurements and high-speed camera imaging, have been mostly centered on printed organic solar cell fabrication.2020. S. Kommeren, M. J. J. Coenen, T. M. Eggenhuisen, T. M. W. L. Slaats, H. Gorter, and P. Groen, Org. Electron. 61, 282 (2018). https://doi.org/10.1016/j.orgel.2018.06.004 Moreover, it is possible to obtain a highly conductive inkjet printable PEDOT:PSS in combination with an ionic liquid additive, as shown in elastic interconnect fabrication.2121. U. Kraft, F. Molina-Lopez, D. Son, Z. Bao, and B. Murmann, Adv. Electron. Mater. 6, 1900681 (2020). https://doi.org/10.1002/aelm.201900681 PEDOT:PSS formulations have been used in the printing of textile and paper-based electrophysiology electrodes, for cutaneous sensing. By employing ethylene glycol, organic solvents and a surfactant, the jetting and drying processes of the ink were optimized on such unconventional substrates.22,2322. E. Bihar, T. Roberts, E. Ismailova, M. Saadaoui, M. Isik, A. Sanchez-Sanchez, D. Mecerreyes, T. Hervé, J. B. De Graaf, and G. G. Malliaras, Adv. Mater. Technol. 2, 1600251 (2017). https://doi.org/10.1002/admt.20160025123. E. Bihar, T. Roberts, Y. Zhang, E. Ismailova, T. Hervé, G. G. Malliaras, J. B. De Graaf, S. Inal, and M. Saadaoui, Flexible Printed Electron. 3, 034004 (2018). https://doi.org/10.1088/2058-8585/aadb56 Indeed, the inkjet processing is strongly dependent on substrate surface properties. The role of several printing parameters to improve the PEDOT:PSS ink deposition on hydrophobic silicon surfaces, including drop spacing, substrate temperature, and the number of layers, have been thoroughly investigated for solar cell manufacturing.1919. M. Chen, A. Iyer, and R. Opila, in IEEE 46th Photovoltaic Specialists Conference (PVSC), 2019. Therefore, similar studies would be necessary to move this patterning technique from silicon-based, planar flexible devices to novel fields of applications.With an increase in the adoption of printable materials in the field of bioelectronics and wearable devices, biocompatibility, mechanical robustness, and environmental stability evaluations are often necessary to justify the choice of materials. Several studies demonstrated promising compatibility of commercially available PEDOT:PSS inks with cell cultures,24–2724. S. Stříteský, A. Marková, J. Víteček, E. Šafaříková, M. Hrabal, L. Kubáč, L. Kubala, M. Weiter, and M. Vala, J. Biomed. Mater. Res., Part A 106, 1121 (2018). https://doi.org/10.1002/jbm.a.3631425. L. D. Garma, L. M. Ferrari, P. Scognamiglio, F. Greco, and F. Santoro, Lab Chip 19, 3776 (2019). https://doi.org/10.1039/C9LC00636B26. M. Solazzo, K. Krukiewicz, A. Zhussupbekova, K. Fleischer, M. J. Biggs, and M. G. Monaghan, J. Mater. Chem. B 7, 4811 (2019). https://doi.org/10.1039/C9TB01028A27. M. Sessolo, D. Khodagholy, J. Rivnay, F. Maddalena, M. Gleyzes, E. Steidl, B. Buisson, and G. G. Malliaras, Adv. Mater. 25, 2135 (2013). https://doi.org/10.1002/adma.201204322 cutaneous contact,28,2928. L. M. Ferrari, U. Ismailov, F. Greco, and E. Ismailova, Adv. Mater. Interfaces 8, 2100352 (2021). https://doi.org/10.1002/admi.20210035229. L. M. Ferrari, S. Sudha, S. Tarantino, R. Esposti, F. Bolzoni, P. Cavallari, C. Cipriani, V. Mattoli, and F. Greco, Adv. Sci. 5, 1700771 (2018). https://doi.org/10.1002/advs.201700771 and even in vivo neural interfacing.3030. Y. Zhou, B. Ji, M. Wang, K. Zhang, S. Huangfu, H. Feng, H. Chang, and X. Yuan, Coatings 11, 204 (2021). https://doi.org/10.3390/coatings11020204 In wearables, adverse reactions can occur at the interface with the body such as tissue irritation, inflammation, or development of a foreign-body response. Concerning synthetic reactivity, organic materials and flexible devices that are composed of carbon are further envisioned for green processing and efficient recycling.3131. M. He, Y. Sun, and B. Han, Angew. Chem., Int. Ed. 61, e202112835 (2022). https://doi.org/10.1002/anie.202112835 According to chemical datasheets, some components can be considered as non-hazardous, taking into account their exposure levels vis a vis contact with skin, eyes, inhalation, ingestion, acute and chronic contact, and their dosage. A key aspect is to characterize their potential biocompatibility, which requires validation prior to a particular usage scenario. Therefore, this practice is guided by the use of non-hazardous chemicals when bio-interfacing, as well as taking into account the sustainability aspects of manufacturing. The use of green solvents and reagents during synthesis reduces the quantity of toxic chemical waste generated and allows for non-hazardous disposal.

Here, we present an electrically conducting PEDOT:PSS formulation for the inkjet printing of wearable devices. The ink's electrical properties were evaluated in correlation with the specifics of the printability process. The key characteristics were experimentally determined for a broad printability assessment in relation to the theoretical predictions. The mechanical and water stability results indicate an appropriate robustness of the printed designs for diverse wearable applications. With a view to target wearable bioelectronic device manufacturing, the cytotoxicity assays show high human stem cells viability when in contact with the ink-coated substrates. Finally, the formulated ink allows the fabrication of a printed, wearable gait sensor on a paper-like substrate, with a minimal device footprint while precisely tracking walking activity.

III. DISCUSSION

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ChooseTop of pageABSTRACTI. INTRODUCTIONII. RESULTSIII. DISCUSSION <<IV. CONCLUSIONSV. MATERIALS AND METHODSSUPPLEMENTARY MATERIALThe addition to the DMSO, IPA, TWEEN20 surfactant, and the GOPS cross-linking agent to the PEDOT:PSS dispersion resulted in a performant, printable ink formulation. As reported, the DMSO enhances the electrical conductivity by improving polymeric chains morphological rearrangement during the film drying process.5252. Q. Wei, M. Mukaida, Y. Naitoh, and T. Ishida, Adv. Mater. 25, 2831 (2013). https://doi.org/10.1002/adma.201205158 Indeed, in commercial PEDOT:PSS water dispersion, an excess of PSS chains is added, both to act as a counterion for the PEDOT+ chains and to electrostatically stabilize them in an aqueous suspension. The PSS has an insulating nature; thus, the excess of PSS increases the overall material electrical resistance. DMSO reduces the electrostatic inter-chain interactions between the PEDOT rich grains and the insulating PSS rich shells. In the deposited film, this contribution results in the coarsening of PEDOT domains, which, in turn, results in the formation of more conductive pathways, ultimately facilitating the inter-PEDOT chains' charge transport.53–5553. C. Deetuam, D. Weise, C. Samthong, P. Praserthdam, R. R. Baumann, and A. Somwangthanaroj, J. Appl. Polym. Sci. 132, 42108 (2015). https://doi.org/10.1002/app.4210854. P. Wilson, C. Lekakou, and J. F. Watts, Org. Electron. 13, 409 (2012). https://doi.org/10.1016/j.orgel.2011.11.01155. R. M. Vedovatte, M. C. Saccardo, E. L. Costa, and C. E. Cava, J. Mater. Sci. 31, 317 (2020). https://doi.org/10.1007/s10854-019-02524-1 On the contrary, the IPA's low boiling point solvent controls the so-called “coffee ring effect.” When a drop is dispensed onto a substrate, the solvent's evaporation occurs faster at its edges. Capillary forces then drag the ink toward the drop boundaries, resulting in migration toward the drop boundary. Thus, leading to excess deposition and solidification of the ink solute at the border.1212. H. Hu and R. G. Larson, J. Phys. Chem. B 110, 7090 (2006). https://doi.org/10.1021/jp0609232 The combination of low and high boiling point solvents avoids inhomogeneities in the dried film, changing the shape of solute deposition from a circumferential, ring-like pattern to a solid dot-like shape.3636. B.-J. de Gans and U. S. Schubert, Langmuir 20, 7789 (2004). https://doi.org/10.1021/la049469oThe addition of TWEEN20 was also crucial to optimization of the ink formulation, since surfactants help in improving film homogeneity by reducing the fluid surface tension.3737. S.-S. Yoon and D.-Y. Khang, J. Phys. Chem. C 120, 29525 (2016). https://doi.org/10.1021/acs.jpcc.6b12043 A homogeneous coating occurs when the surface energy of the substrate has a higher value than the surface energy of the solution, yet not too different. In this respect, surfactant molecules reduce the surface tension to a value closer to the surface energy of the substrate, and enhance crystallization of PEDOT chains due to the weakening of the ionic interactions between PEDOT and PSS.3737. S.-S. Yoon and D.-Y. Khang, J. Phys. Chem. C 120, 29525 (2016). https://doi.org/10.1021/acs.jpcc.6b12043 By depositing defined patterns with appreciable qualities onto different substrates, the beneficial role of the aforementioned additives throughout the full printing process was demonstrated. Additionally, it allowed for optimal inkjet printing parameters to be defined.A performant ink must be stable under air exposure, high humidity circumstances, and long-term water immersion with negligible conductivity decay or structure deterioration.5656. Y. Wen and J. Xu, J. Polym. Sci., Part A 55, 1121 (2017). https://doi.org/10.1002/pola.28482 Therefore, we investigated the conductivity of the printed film with respect to its thickness and then assessed the film's flexibility in terms of bending capability as well as stability in aqueous environment. The mechanical cycling stress tests conducted on the ink revealed appreciable intrinsic electrical stability under tension strain, a highly required characteristic in flexible electronics applications. This means that an excellent film adhesion is developed at the interface with the substrate. Additionally, the deposited ink layers showed adequate water stability over time, exhibiting good retention of electrical properties and appreciable adhesion to the substrate under wet conditions.Therefore, the combination of inkjet printing, which has been reported as a deposition method that enhances the PEDOT:PSS films' water stability,2525. L. D. Garma, L. M. Ferrari, P. Scognamiglio, F. Greco, and F. Santoro, Lab Chip 19, 3776 (2019). https://doi.org/10.1039/C9LC00636B together with GOPS, represents a promising approach to characterize the film properties lost in water over time in wearable conditions. Overall, both the impedance and the film transmittance confirm the stability of our ink in contact with water. Remarkably, the interplay of the employed additives play a role in controlling and enhancing the ink performance. In addition, their nontoxic nature confers the material with promising qualities for bio-interfaced device fabrication. Indeed, the preliminary cytocompatibility evaluations do not reveal any noxious effects on human mesenchymal stem cells, yet not fully implying its non-hazardless. Further experiments will be required to assess the complete biocompatibility of the fabricated devices in accordance with specific regulations. Finally, the ink deposition on off-the-shelf melt-blown fabric resulted in an effective method for a one-step gait sensor fabrication, which is directly transferable to a variety of substrates according to the application needs. The embedding of soft conducting materials onto fabric is an established approach to obtain electronic textiles in the shape of smart garments employed for a variety of physical and biochemical body parameter monitoring.22,57,5822. E. Bihar, T. Roberts, E. Ismailova, M. Saadaoui, M. Isik, A. Sanchez-Sanchez, D. Mecerreyes, T. Hervé, J. B. De Graaf, and G. G. Malliaras, Adv. Mater. Technol. 2, 1600251 (2017). https://doi.org/10.1002/admt.20160025157. S. Cho, T. Chang, T. Yu, and C. H. Lee, Biosensors 12, 222 (2022). https://doi.org/10.3390/bios1204022258. S. Takamatsu, T. Lonjaret, D. Crisp, J.-M. Badier, G. G. Malliaras, and E. Ismailova, Sci. Rep. 5, 15003 (2015). https://doi.org/10.1038/srep15003 The sensor shows great potential to distinguish between slow and fast walking rates, providing information on the user's walking activity and insights on the plantar pressure distribution. Such spatial information can be translated, after extended calibration, into a foot pressure map.

IV. CONCLUSIONS

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ChooseTop of pageABSTRACTI. INTRODUCTIONII. RESULTSIII. DISCUSSIONIV. CONCLUSIONS <<V. MATERIALS AND METHODSSUPPLEMENTARY MATERIAL

We have presented an effective methodology to formulate and characterize an inkjet printable PEDOT:PSS formulation from a commercial solution all the way to a wearable device fabrication. We theoretically evaluated the ink's printability and showed experimentally high-quality ink deposition on some of the most used substrates in bio- and flexible electronics. We show here how to balance and optimize the interactions between the materials and the inkjet printing process. Indeed, the ink's chemical composition resulted in a material with enhanced electrical properties, mechanical flexibility, and water stability that are particularly interesting for wearable bioelectronic devices. The ink formulation approach can be easily translated to any water-soluble conjugated polymers. The use of known chemicals resulted in an ink that is cytocompatible. The wearable step tracker, fabricated through the patterning of the formulated ink onto a paper-like fabric substrate, shows great potential to be seamlessly integrated into wearables, such as shoes or socks. The developed ink offers high applicability and versatility in disposable electronics, conformable biomedical devices, and green flexible sensors.

V. MATERIALS AND METHODS

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. RESULTSIII. DISCUSSIONIV. CONCLUSIONSV. MATERIALS AND METHODS <<SUPPLEMENTARY MATERIAL

A. Ink formulation

The ink consists of commercially available PEDOT:PSS water dispersion Clevios PH1000 (Heraeus) 10% w/w of DMSO (Merck), 5% w/w of IPA (Merck), and 0.5% w/w of TWEEN20 (Merck). The solution was mixed for 20 min in an ultrasonic bath. The ink was kept in the fridge at 4 °C. Just before printing, 1% w/w of (3-glycidyloxypropyl)trimethoxysilane GOPS (Merck) was added.

B. Ink rheology

The surface tension of the solutions is evaluated at constant ambient temperature with an optical contact angle measuring unit and a contour analysis system (Apollo Instrument, OCA200) via the pendant drop method. The same tool was used to measure the solution's contact angle on a glass substrate. The ink viscosity was measured by scanning the shear rate from 1 to 1233 s−1, and the actual value was taken at 1000 s−1.5959. R. M. Meixner, D. Cibis, K. Krueger, and H. Goebel, Microsyst. Technol. 14, 1137 (2008). https://doi.org/10.1007/s00542-008-0639-7

C. Droplets characteristics and resolution

Prior to printing, the ink was filtered with a 0.2 μm cellulose acetate filter. We employed Dimatix Fujifilm DMP 2800 Inkjet printer (Dimatix Material Printer, Fujifilm Dimatix, Santa ovenClara, CA, USA) with 10 and 2.4 pl nominal drop volume cartridges.

The flexible substrates used for characterization were parylene C vapor deposited film (SCS Labcoter), polyimide Kapton foils (ADDEV Materials), temporary tattoo paper (Silhouette America, Inc, US), and a commercial non-woven fabric. Before printing, only parylene C and Kapton substrates were surface-treated, through a mild O2 plasma treatment at 50 W for 1 min (PE100—Plasma Etch, Inc). We performed the ink curing process, of those printed on the parylene C and Kapton samples by placing them on a hot plate at 130 °C for 10 min, while we cured the tattoo paper and fabric samples in an oven at 60 °C for 1h. Before any further characterization, we washed the samples with de-ionized water to remove the excess of PSS chains, potentially not-crosslinked molecules and unreacted GOPS fraction, and then dried the samples with nitrogen gas. To investigate the achievable resolution, we inspected the resolution patterns printed on different substrates through images obtained with an optical microscope (Nikon Eclipse L200).

D. PEDOT:PSS film characterization

1. Electrical properties

To electrically characterize the ink, we printed one to multiple layers of a squared design (area 1 cm2) on a polyimide Kapton substrate. We measured the film thickness with a mechanical profilometer (AMBIOS technology XP-2) and the sheet resistance through a four-point set-up (Keithley source measure unit).

2. Mechanical properties

We assessed the formulated ink performance while undergoing dynamic bending stress with a push to flex bending setup. For this characterization, samples were prepared as follows: Stripes with the size of 50 × 5 mm2 made of one layer of PEDOT:PSS ink were printed on a thin parylene C film (10 μm thick) supporting substrate to minimize the substrate's mechanical properties impact on the experiments' outcomes; then the ink was cured on a hot plate at 130 °C for 10 min. The test sample was clamped at its extremities to two plates; its flat position was set as the position 0. Then the plates were moved closer until the sample was bent with a curvature radius of 0.8 mm, which was set as the position 1. A bending cycle consists in moving the plates back and forward between 0 and 1 position. For each sample, we performed 5000 bending cycles and monitored the sheet resistance of the PEDOT:PSS printed film connecting electrically the sample to a Keithley source measure unit. In this setup, schematized in Fig. S4, the conductive PEDOT:PSS film underwent elastic tension strain.

3. Stability in water

For the electrochemical impedance spectroscopy (EIS), we immersed 1 cm2 printed glass samples in a phosphate buffered saline (PSB) connecting it to the working electrode of a potentiostat (Metrohm Autolab, Nova 2.1). As the reference and counter electrodes, we used Ag/AgCl and a platinum wire, respectively. We computed the impedance with the potentiostatic mode (0.01 V, 0.1–100 kHz).We evaluated the ink's water stability by monitoring the dry film transmittance with a spectrophotometer (Shimadzu UV-2600) after storing the samples in de-ionized water for 16 days and vacuum-dried it before performing measurements.

4. Cytocompatibility assay

Cell culture: Human mesenchymal stem cells (hMSCs, Lonza, Switzerland) were expanded in a T75-flask to passages 5 (Presto Blue assay) and 7 (live/dead assay from Thermo Fisher Inc.) in α-MEM medium (Sigma) supplemented with 10 vol. % fetal bovine serum (Gibco), 1 vol. % glutamine (Glutamax Gibco), and 1 vol. % penicillin/streptomycin (Sigma). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2, and the medium was changed twice a week. Before confluence, the hMSCs were seeded at a density of 6200 cells/cm2 on the materials (previously sterilized with 70% ethanol for 15 min) inside the wells of a 24-well plate. Tested materials were ink-coated glass coverslips (1 cm). The ink was deposited by drop casting, and it was cured at 130 °C for 15 min. The positive control consisted of dead cells, was produced by seeding cells on the glass substrate and further treating them with 70% methanol (in media as recommended by the manufacturer) for 30 min just before performing the Presto Blue or the live/dead assay. The negative control (all cells alive) consisted of the same seeded glass substrate, but without the methanol treatment. Materials were assessed in triplicate (n = 3).

Metabolic activity: Before the assay, materials were moved to a new 24-well plate. Presto Blue reagent (Invitrogen) was then added to the wells at a concentration of 10 vol. % after 1, 3, and 7 days of culture. Fluorescence was measured (Fluoroskan Ascent, Thermo Scientific) at 590 nm (excitation 530 nm). Fluorescence intensity obtained for the samples was then corrected by subtracting the fluorescence obtained from the equivalent blank controls (same materials cultured under the same conditions, but no cells were seeded on them). Statistical analyses were performed with Jamovi software. Comparisons between materials were made with the non-parametric Student's t-test.

Live/dead imaging: A staining solution containing 2 μM ethidium homodimère III (Interchim), 4 μM calcein AM (Molecular Probe), and 1 μg/ml Hoechst (Thermo Scientific) in PBS (Sigma) was added to the culture wells. After 40 min of incubation, cells were rinsed three times with PBS and further observed under a fluorescence microscope (Zeiss Axio Vert.A1). Excitation wavelengths were 385, 475, and 555 nm for Hoechst, calcein, and ethidium stains, respectively.

E. Printed wearable step tracker

1. Sensor fabrication

The wearable sensor was fabricated by printing five layers of the digitally drawn design onto commercial melt-blown fabric, considered as a non-woven fabric that has similarity with compact fibrillar texture of paper structure, without any substrate conditioning. The sensor was placed in an oven at 60 °C for one hour to allow for the complete evaporation of the ink's solvents. The electrical performances of different sensor layouts were investigated with a multimeter. The fabrication process can be replicated on different paper-like substrates including papers destined for recycling.

2. Sensor performances

The printed sensor's performance was investigated by facing two equal serpentines electrodes, one in front of each other. Each of the electrodes was electrically connected to an electrical source measurement unit (National Instruments USB-6251 BNC) through oval-shaped interconnections, see Fig. 3(a). The measurement unit recorded the current passing through the two serpentine electrodes as the output signal of the sensor's system. To simulate the gait, we placed a weight (3 kg) onto and away the sensor at three frequencies (0.5, 0.67 , and 0.83 Hz). The weight laying onto the sensor simulated the pressure occurring when the foot touched the ground, while the weight being lifted simulated the foot lifted from the ground during the gait.