{"paper_id":"24c27ebd-8ed3-43d2-862c-7cb7fab606b5","body_text":"1 \n \nNanoporous Microelectrodes for Neural Electrophysiology Recordings in Organotypic \nCulture  \n \nPetro Lutsyk*, Debjani Goswami, Stuart D. Greenhill* \n \n \nP. Lutsyk, D. Goswami \nAston Institute of Photonic Technologies, College of Engineering and Physical Sciences, Aston \nUniversity, Aston Triangle, B4 7ET, Birmingham, UK \nE-mail: p.lutsyk@aston.ac.uk \n \nS. D. Greenhill \nAston Institute of Health and Neurodevelopment, College of Health and Life Sciences, Aston \nUniversity, Aston Triangle, B4 7ET, Birmingham, UK \nE-mail: s.greenhill@aston.ac.uk \n \n \nFunding: British Academy, Royal Academy of Engineering and Royal Society (Academies \nPartnership in Supporting Excellence in Cross-disciplinary research award - APEX award, \nAA21\\100133 APEX Awards 2022). \n \n \nKeywords: electrophysiology, organotypic culture, microelectrodes, inkjet printing, aerosol jet \nprinting. \n \n \n \nAbstract \nOrganotypic cultures, specifically brain slices, have been used in neuroscience studies for many \nyears to prolong the lifetime of the biological tissue outside of the host organism. However, the \ncultures must be kept in a sterile environment, maintaining supply of gas/nutrients for tissue \nsurvival and physiological relevance. Electrophysiological recordings from cultured tissue are \nchallenging as the conventional approaches implicate a compromise on biological stability or \nenvironmental integrity. In this article, a novel approach has been used to design and print \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n2 \n \nnanoporous microelectrodes on culture wells enabling in situ recording of electrophysiological \nneural activities. Optimized ink formulations are developed for conductive nanocarbon \nmicroelectrodes, and furthermore, fluoropolymer (polytetrafluoroethylene-based AF2400) ink has \nbeen inkjet printed for the first time acting as an insulator layer for microelectrodes. To keep the \nbiocompatible nanoporous structure of culture wells, the microelectrodes have been printed on the \nbottom of the culture cells and only small connector pads have been produced on top of the \nculture membrane. Neural activity has been recorded by such a microelectrode structure for rodent \nbrain slices cultured for three weeks. Furthermore, aerosol jet printing has been used for printing \nof nanocarbon microelectrodes allowing to produce much smaller size features compared to the \ninkjet printing. \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n3 \n \n1. Introduction \n \nCultured tissues have been used in biological research for many years and are also experiencing a \nresurgence due to an increasing application of organoids & other multicellular systems. [1,2] \nOrganotypic cultures of brain slices are utilized by neuroscientists to prolong the lifetime of such \ntissue outside of the host organism and obtain valuable electrophysiological recordings in vitro, \nfor example, epileptic human tissue obtained through brain resection. [3] Extracting meaningful \nfunctional data from cultured slices is challenging due to the need to compromise the sterile \nenvironment of cultures or to remove the culture into a sub-optimal environment to facilitate \nelectrophysiological recordings. Organotypic culture techniques allow preservation of tissue slices \nin as close to their natural state as possible for weeks, providing an opportunity for long-term \nexperimental manipulation, drug dosing, and other interventions. However, the cultures must be \nkept in a sterile environment, maintaining proper supply of gas/nutrients for tissue viability.  \nOne of the ways to preserve the culture environment for electrophysiology recordings is to \nintegrate the microelectrodes or microelectrode array (MEA) into culture wells. However, \nconventional MEAs are fabricated on rigid substrates, and metals are used for electrodes. [4] The \nmicroelectrodes for culture substrate integration need to be biocompatible and porous to replicate \nthe culture well and allow nutrients/gas to access the tissue. Novel materials for microelectrodes \nhave been widely researched, and several studies are confirming that graphene-based MEAs are \nan excellent candidate for neural recordings due to their biocompatibility, flexibility, and \nexceptional electrical properties. [5,6] Viana et al recently demonstrated the feasibility of graphene-\nbased microelectrodes for in vivo brain recordings with high fidelity and superior implantation \nbiocompatibility. [7] The electrode material is recognized as a critical factor that determines the \nperformance of neural interfaces in such implants. [7,8] Furthermore, neural cell cultures on \ngraphene substrates showed good viability with enhanced neurite growth. [9] Another outstanding \nfeature of graphene is its ability to form structures with high double layer capacitance at the \ninterface of conductive microelectrodes with electrolyte (i.e., aqueous media). [7,8] Porous \ngraphene electrodes have been explored aiming to overcome the challenging trade-off between \nporosity and electrode performance. [7,10,11] To the best of our knowledge, there are no studies of \nintegrating media-permeable nanoporous microelectrodes to culture well substrates, enabling \ncontinuous monitoring of neural activity of organotypic cultures.  \nIn this paper, the innovative approach has been used to design and print microelectrodes on \nhighly porous culture wells allowing to record electrophysiological neural activity for rodent brain \nslices. To keep the biocompatible nanoporous structure of culture wells, the microelectrodes were \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n4 \n \ndouble side printed on the culture substrates with insulative layers on the bottom and only a small \nconnector pad contacting cultured tissue on the top (Figure 1). Designed microelectrode structure \nenabled neural recordings for the tissues cultured for three weeks. Optimized ink formulations \nwere developed for conductive nanocarbon microelectrodes and insulative polymer layers. \nImportantly, inkjet printing of PTFE-based polymer layers has been demonstrated for the first \ntime. The presence of the insulative layer for microelectrodes was a key factor in obtaining good \nquality recordings, as the microelectrodes without insulative layers (Figure 1a,b) had substantial \ndissipation of the electrophysiology signal to the fluids preventing to record electrical activities of \norganotypic cultures. \n \n \n \nFigure 1. (a-c) Schematic architecture of the microelectrodes on the membrane for organotypic \nculture, where (a) microelectrodes were printed only on top of the membrane, (b) double side \nmicroelectrodes (b) without and (c) with insulator at the bottom of the membrane; (d) nanocarbon \nink drop produced from 50 µm nozzle; (e) inkjet printing setup with 30mm Millicell culture plate \ninsert; (f) cultured brain tissue in the culture well with microelectrodes printed as in the part (c). \n \n \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n5 \n \n2. Results and Discussions \n \n2.1. Conductive Ink Formulation \nConductive nanocarbon microelectrodes have been produced by application of shear-force mixing \nwith 1-pyrenesulfonic acid (PS1) as surfactant. Pyrene sulphonic derivatives are proven to be one \nof the most efficient surfactants in producing water-based stable (up to 1 year) dispersions of \ngraphene and graphene-based nanocarbon. [12,13] Such dispersions yield high concentrations of \nnanocarbon without oxygen groups [12] and are shown to be inkjet printable [13] even considering \nthat their inverse Ohnesorge number Z is higher than 14. Z is expected to be in the range of 1 < Z \n< 14 to produce stable drops. [14] For inkjet printer settings with nozzle diameter (d) = 50 µm, \nwater-based ink would produce Z ~ 60, much exceeding the above range. Therefore, additional \nrefinements for nanocarbon ink formulations were required. Adding Triton X-100 allowed us to \nreduce surface tension from 72.8 mN/m (water) to about 33 mN/m. Propylene glycol admixed to \nour printing solution resulted in the viscosity increasing up to 2 mPa·s. Due to the reduced surface \ntension and increased viscosity, our nanocarbon ink has Z ~ 20 for a nozzle diameter of 50 µm \n(Table S1). Such ink produced a stable drop (Figure 1d) even with Z being slightly above 14; a \nsimilar outcome was shown before. [13] Furthermore, the resulting ink had diminished coffee-ring \neffect (due to weaker Marangoni flow [15]). Finally, the obtained nanocarbon ink is suitable for \naerosol jet printing via ultrasonic atomization, which works well for inks with a maximum \nviscosity of 5 mPa·s. [16] \nIt was previously shown that shear-force mixing is a scalable, defect-free, and highly \nefficient technique for graphene exfoliation (alternative to sonication) and, as a proof-of-concept, \nthese studies used sodium cholate as a surfactant to disperse graphene in water. [17,18] Application \nof PS1 for graphene exfoliation required 72 hours of tip sonication to produce highly concentrated \nPS1-based ink. [13] We have applied shear-force mixing with PS1 to produce nanocarbon ink, and \nonly 2 hours of treatment was needed to reach the required level of nanocarbon concentration. \n \n2.2. Insulative Ink Formulation \nFor insulative ink, we have used perfluorodecalin solution of AF2400 (5 mg/mL). The polymer \nwas chosen because of its excellent gas permeability due to the inherent extensive free volume \n[19,20] and having a molecular structure similar to the culture membrane substrate (made of PTFE). \nSurface tension and viscosity of such solution were 27 mN/m and 5.5 mPa·s, respectively (slightly \nincreasing compared to the features of neat perfluorodecalin). This ink formulation has Z = 9.5 for \nd = 50 µm (Table S1), and a stable drop was formed during inkjet printing. Apart from having Z \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n6 \n \nwithin the expected range, polymer-ink printing needs to follow viscoelastic jet stability to avoid \n‘bead-on-a-string’ structure achieved for flexible polymers of sufficiently high molecular weight. \n[21] Therefore, the solution of AF2400 has been tip sonicated, aiming to reduce the molecular \nweight of the polymer and ensure more stable drop formation. \n \n2.3. Ink Characterization by Absorption and Photoluminescent Spectroscopy \nThe nanocarbon concentration in the inks was determined using absorption spectroscopy in \nthe visible range. The absorption spectrum of graphene and larger graphite nanoparticles is nearly \nflat and has no features in the long-wavelength visible region (Figure S1). There are extensive \nstudies in which the absorption is measured at 660 nm for the concentration estimation using the \nBeer–Lambert law. [17,18] The concentration estimation by absorption coefficient for such material \nhas some variance, [22] therefore, in this study a conventional value for absorption coefficient at \n660 nm (α660 = 2460 L g-1 m-1) [23] was used. By this technique, nanocarbon concentration in the \nink was evaluated to be in the range of 1 mg/mL. The nanocarbon ink still has some content of \nPS1, as the characteristic PS1 absorption peak at 375 nm is present in the ink spectrum (Figure \nS1). Insulative ink, perfluorodecalin solution of AF2400, has the absorption maximum of 206 nm \nand is practically transparent in the UV, visible, and near infrared range from 250 nm (Figure S1).  \nPhotoluminescence (PL) measurements demonstrated that the nanocarbon ink is featured by \nmonomer emission of PS1 molecules in the range of 370-430 nm (Figure 2). Such monomer \nemission has characteristic narrow peaks at 374, 393, 413 nm and small spectral bands at 382 and \n403 nm. However, the most significant PL feature of the ink is a lack of broad excimer emission \nof the PS1 in the range of 430-600 nm (peaking at 494 nm) that is present in the highly \nconcentrated aqueous solution of PS1 (Figure 2c, curve 3) and supernatant obtained during the ink \nfabrication (Figure 2c, curve 2). The excimer emission evidences the formation of dimers of PS1 \nmolecules and demonstrates a substantial excess of PS1 surfactant in the supernatant.  \nAnother feature of nanocarbon ink emission is an amplification of PL band at 413 nm and \nthe emergence of a new band at 434 nm and a broad shoulder in the range of 450-475 nm. These \nink features are appearing at excitation wavelength range 340-400 nm (Figure 2a) and can be \nassociated with weak emission of nanocarbon particles in the dispersion. \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n7 \n \n350 400 450 500 550 600\n300\n325\n350\n375\n400\n425\n450\nink\nExcitation wavelength, nm\nEmission wavelength, nm\n0\n1×106\n2×106\n3×106\n4×106\n5×106\n6×106\n7×106\n8×106\n(a)\n \n350 400 450 500 550 600\n300\n325\n350\n375\n400\n425\n450\nsupernatant\nExcitation wavelength, nm\nEmission wavelength, nm \n0\n1×106\n2×106\n3×106\n4×106\n5×106\n6×106\n7×106\n(b)  \n350 400 450 500 550 600\n0\n1×106\n2×106\n1.  ink\n2.  supernatant\n3.  PS1\nPL intensity, cps\nWavelength, nm                                                                                               \n(c)\n \n350 400 450 500 550 600\n0\n1×106\n2×106\n1.  ink\n2.  supernatant\nPL intensity, cps\nWavelength, nm                                                                                               \n(d)\n1 2  \nFigure 2. (a,b) Excitation-emission photoluminescence (PL) maps of nanocarbon ink (a) and \nsupernatant obtained in the process of ink formulation. (c,d) PL spectra of nanocarbon ink (c,d - \ncurves 1) supernatant obtained in the process of ink formulation (c,d – curves 2); and aqueous \nsolution of PS1 at the concentration of 6 g/L used in the ink formulation (c – curve 3); \nexcitation wavelengths are 310 (c) and 375 (d) nm. \n \n \n2.4. Inkjet and Aerosol Printed Microelectrodes \nThe hydrophilic PTFE membrane of Millipore culture inserts with 0.4 µm pores (according to the \nspecs) has a highly branched network of polymer micro-sized wires connected to multiple nodes, \nand this is well evidenced by our SEM measurements (Figure 3a). The multi-node, mesh structure \nof the PTFE substrates should be responsible for high viability (for as long as 40 days) and \nexcellent trans-membrane oxygen transport. [24] Therefore, the porous features of such membranes \nneed to be preserved during the microelectrode’s integration to such substrates. \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n8 \n \n  \n  \nFigure 3. SEM images for (a) neat PTFE membrane of culture insert substrate, (b) aerosol jet \nprinted nanocarbon microelectrode on the PTFE membrane; (c) aerosol jet printed microelectrode \n(bottom) with AF2400 insulative cover (middle) on the PTFE membrane (top); (d) inkjet printed \nmicroelectrode (middle dark area) with AF2400 insulative cover on the PTFE membrane. \n \n \nAerosol jet and inkjet printing of the formulated in this study inks resulted in a good quality \nmicroelectrode/insulator lines (Figure 3b,c). The width of the aerosol jet printed microelectrode \n(20 printing passes having ca. 1MΩ resistance) is approx. 45 µm, whereas inkjet printed \nmicroelectrode width (10 printing passes - ca. 1MΩ resistance) extends up to approx. 100 µm. \nThus, aerosol jet printing for nanocarbon microelectrodes allowed us to produce much smaller \nsize features compared to the inkjet printing. Besides, the edges of the aerosol jet printed \nmicroelectrodes (Figure 3b) are smoother than the inkjet printed lines (Figure 3d) due to a stable \naerosol jet with little or no overspray (minimizing overspray is identified as one of the major \nchallenges in the initial process development for aerosol jet printing [16]). It needs to be noted that \nthe conductivity of the microelectrodes could be improved by thermal treatment, but such a step \nhas been eliminated at this stage of the studies to avoid additional impact on the biocompatibility \n(a) (b) \n(c) (d) \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n9 \n \nof Millipore culture insert membranes. Furthermore, nanocarbon ink formulation can be further \noptimized, or an alternative treatment of microelectrodes applied to improve the electrical \nproperties of printed lines. \nInkjet printing of AF2400 ink resulted in the formation of widened insulation lines of \napprox. 200 µm (for 20 printing passes). Wider lines of AF2400 ink are formed due to a high \nwettability of perfluorodecalin-based ink with the PTFE membrane (Figure S2-S4). Inkjet printing \nof insulator lines ‘sandwiching’ conductive lines works very well for aerosol jet printing of \nmicroelectrodes, allowing ample offset to cover a 45-µm-width of the nanocarbon conductor by a \n200-µm-width AF2400 insulator. Initial biocompatibility studies showed that a high number (>40) \nof printing passes for insulative ink resulted in a filling of the PTFE nanopores and the \nmembranes losing permeability feature. Therefore, too much of AF2400 insulative cover either on \ntop or at the bottom of the PTFE membrane significantly reduces the viability of the organotypic \ncultures due to reduced trans-membrane porosity and subsequent supply of media \nsupplements/O2/CO2. Aiming to achieve reasonable insulation but preserving the nanoporous \nstructure of the culture insert substrate, AF2400 insulative cover required thickness optimization, \nwhere having 20 printing passes provided good outcomes with a trade-off between \nbiocompatibility and insulation. SEM images (Figure 3c) demonstrate that the areas of AF2400 \ninsulative cover still have pores, but the branched network has wider PTFE membrane wires being \ncovered by AF2400. \nAF2400 and nanocarbon prints have an excellent adhesion to the PTFE membranes, as well \nas nanocarbon prints on PTFE covered by AF2400. Prolonged keeping of the microelectrodes in \nthe media environment did not result in any noticeable deterioration of the microelectrode \nphysical appearance and conductivity/resistance. Importantly, this evidences a feasibility of the \ntechnology for producing stable conductive microelectrodes/insulative layers on the culture \nsubstrates. \nThe initial design of microelectrodes was to print all the structures on top of the membrane \n(Figure 1a). However, the signal from such microelectrodes without insulative layer was very low, \nwhereas adding even very thin insulation on top of the membrane resulted in a compromised \nbiocompatibility. Therefore, to maintain the nanoporous structure of culture wells biocompatible, \ndouble side printing of microelectrodes has been considered (Figure 1b), where most of the \nprinting is happening at the bottom and only small connector pads are designed to be on top of the \nsubstrate. The double side printing without insulative layer covering microelectrodes (Figure 1b), \nsame way as schematics on top of the membrane only (Figure 1a), resulted in substantial \ndissipation of the electrophysiology signal to the conductive media of organotypic cultures \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n10 \n \ninhibiting registration of the electrical pulses. Thus, the double side microelectrodes with \ninsulative layer (at the bottom of the substrate) covering most of the conductive parts have been \ndesigned (Figure 1c). Such schematics leave uncovered only a small connector pad on top of the \nculture membrane that connects to the brain slice and another connector pad on the opposite side \nof the microelectrode that is used for connection to the signal registration system. The \nmicroelectrodes, as shown in Figure 1c, appeared to be highly efficient in recording \nelectrophysiology signals with a high signal-to-noise ratio and good biocompatibility. \nPL studies for printed microelectrodes allowed us to understand additional features of such \nsystems. As-printed microelectrodes have distinctive emission peaks at 375, 393, and 413 nm with \nexcitation maximum at 345 nm (Figure 4a). These intense peaks evidence a significant presence \nof PS1 molecules on the substrate with as-printed lines. It needs to be noted that PS1 is spreading \non the PTFE membrane due to the wetting process, particularly for multiple printing passes. For \nnanocarbon ink, the carbon particles (a few hundred nm in size) stay in the place of the ink drop \nlanding on the surface, whereas water (and consequently water-soluble surfactant molecules) can \npenetrate the hydrophilic PTFE membrane and spread further. Such a process becomes visible \nwith the naked eye after several printing passes made in a very short time, as the membrane \naround the printed microelectrode changes color (from non-transparent white to pale translucent). \nAs water gradually evaporates, the membrane around the microelectrode returns to non-\ntransparent white with a slight staining of grey.  \n \n350 375 400 425 450 475 500 525\n300\n325\n350\n375\n400\n0.0\n2.0×105\n4.0×105\n6.0×105\n8.0×105\n1.0×106\n1.2×106\n1.4×106\n1.6×106\n1.8×106microelectrodes on PTFE\nas-printed\nExcitation wavelength, nm\nEmission wavelength, nm\n(a)\n350 375 400 425 450 475 500 525\n300\n325\n350\n375\n400\n0.0\n2.0×105\n4.0×105\n6.0×105\n8.0×105\n1.0×106\n1.2×106\n1.4×106\n1.6×106\n1.8×106microelectrodes on PTFE\nwashed in water\nExcitation wavelength, nm\nEmission wavelength, nm\n(b)\n \nFigure 4. Excitation-emission photoluminescence (PL) maps of the microelectrodes (a) as printed \nand (b) after washing in water (to remove excess PS1). \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n11 \n \nPS1 is well soluble in water, and the follow-up washing of the microelectrodes in water has \nresulted in disappearance of PS1 peaks (Figure 4). It evidences efficient removal of the PS1 \nmolecules from printed microelectrodes. Removing PS1 from microelectrodes allowed us to \nobserve a low-intensity broad PL band having a maximum in the range of excitation wavelengths \n380-400 nm and emission wavelength 450-600 nm that features well a neat PTFE membrane of \nthe Millicell culture plate inserts (Figure S5). In this broad band, there is a potential contribution \nof the low-intensity PL shoulder from nanocarbon, evidenced in Figure 2a (PL emission in the \nrange of 450-475 nm at excitation wavelength range 340-400 nm). Low intensity broad emission \nfrom nanocarbon microelectrodes (on PTFE membrane) provides a good background for potential \nin situ PL studies of organotypic cultures (e.g., simultaneous optical imaging and \nelectrophysiological recording [25]). \n \n \n2.5. Electrophysiology Recordings and Histology \nAfter 21 days in culture using both insulated and non-insulated nanocarbon-printed culture wells, \nelectrophysiological and histological analysis were carried out on the brain slices to assess cellular \nand network function, and to confirm biocompatibility of the printed electrodes. To provoke \nwidespread activity, the GABAA antagonist gabazine (20µM) was added to recordings in order to \ndisinhibit the slice. \nAs would be expected, the clearest and most reliable recordings were obtained from wells, \nwhich had insulating polymer encapsulating the printed nanocarbon traces (Figure 5a,b). With \nexposed nanocarbon traces, short-circuiting of the neuronal signals into the bath aCSF, and too-\nlow impedances reduced the quality and reliability of the recordings. With insulated traces, \ncultured slices were able to be aligned with the recording pads to ensure that identifiable \nanatomical landmarks (e.g., CA1 of the hippocampus) were located on top of the recording site. \nPlacing either glass or solid electrodes on the relevant recording pad resulted in a multi-unit \nrecording of neuronal action potentials that was not evident when the electrode was not directly \nmaking contact with the pad.  \nCultured slices were also fixed and stained with hematoxylin and eosin to allow for \nquantification of cell density. In a 100x100µm region of interest (ROI), there were no significant \ndifferences between control cultures, exposed nanocarbon and insulated nanocarbon (n = 6 per \ngroup, Kruksal-Wallis test) suggesting that any cell loss was consistent across all the culture well \nconstruction, with no biocompatibility concerns from the printed additions (Figure 5c). \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n12 \n \n \n \n \n \nFigure 5. Electrophysiology and biocompatibility of novel culture system. (a) High-impedance \nglass microelectrode recordings show well-defined multi-unit activity in a cultured brain slice in \nthe presence of gabazine. (b) Low-impedance wire electrode recordings also show multi-unit \nactivity with some rebound shot noise. (c) Cell counts show no significant difference in histology \nbetween control, nanocarbon and insulated nanocarbon conditions. (d) Example pad configuration \n(left) and brain slice in situ (right). \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n13 \n \n \n3. Conclusions \nNovel microelectrodes printed on highly porous culture substrates enabled the recording of multi-\nunit neural activity of rodent brain slices cultured for three weeks. The high-quality recordings \nand biocompatible structure of culture membranes were obtained by double side printing of \nmicroelectrodes with the addition of insulative layers. Besides, aerosol jet printing of small-sized \nmicroelectrodes ‘sandwiched’ by inkjet jet printing of insulator lines resulted in the structures with \nsubstantially improved signal-to-noise ratio of electrophysiological recordings. Designing \nnanoporous microelectrodes on culture wells can pave the way for the development of more \nadvanced setups for long-term monitoring of cultured tissues, enabling transformative studies of \nbrain activities & potential for novel treatment of brain pathologies, including personalized \nmedicine. Besides, this technology can drive the reduction in animal use by increasing the \nusability of tissue taken from animals. Finally, the feasibility of inkjet printing for PTFE-based \npolymer has been demonstrated, and this particular outcome can be translated into various \napplications beyond insulating layers for microelectrodes, for example, tailorable optoelectronics \nencapsulation structures, optical and microwave windows.  \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n14 \n \n \n4. Experimental Section / Methods \n \nConductive Ink Preparation. Graphite powder (4g; <20 μm initial size of powder particles; \nSigma Aldrich / Merck) was dispersed in the presence of a preliminary dissolved 1-pyrenesulfonic \nacid (PS1) (240mg) in 40 mL deionized water. An L4RT high shear mixer (Silverson) was used \nfor dispersion with an emulsion screen at 10,200 rpm and at room temperature sample cooling \nbath. The obtained dispersion was centrifuged with a Mini Spin plus centrifuge (Eppendorf SE) at \n3,700 rpm for 20 min, and the top 50% of supernatant was collected and centrifuged again at \n14,200 rpm for 1 hour. Approximately 0.5 mL of precipitate was collected and mixed with 2 mL \nof a printing solution to obtain the nanocarbon ink. The printing solution was obtained by mixing \n2 mL of propylene glycol with 10 mL of deionized water and ≥1 μL of Triton X-100 (all Sigma \nAldrich / Merck). The nanocarbon ink was filtered using a 5-µm-pore PTFE syringe filter.  \n \nInsulative Ink Preparation. Poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-\ntetrafluoroethylene] or short AF2400 was dissolved in perfluorodecalin (both Sigma Aldrich / \nMerck) with the concentration of 5 mg/mL. The solution was ultrasonicated for 20 min and \nfiltered via a 5-µm-pore PTFE syringe filter to ensure polymer-based ink forms good quality \ndrops for inkjet printing. \n \nInk Characterization. The surface tension and contact angle of the ink were obtained with a \nL2004A1 Ossila contact angle goniometer, whereas the viscosity was measured via a falling ball \nviscometer. The absorption spectra were measured with a double beam Lambda 1050 \nUV/VIS/NIR spectrometer (Perkin Elmer) using 3mm or 10mm quartz cuvettes. The \nphotoluminescence spectra and excitation-emission photoluminescence maps were measured by a \nNanoLog excitation–emission spectrofluorometer (Horiba) with high concentration liquid samples \nand solid samples placed in frontal mode at 30 degrees.  \n \nInkjet Printing. Inkjet printing was made using an Autodrop Professional AD-P-8000 with a 50 \nμm nozzle diameter microdispenser head MD-K-130 (Microdrop Technologies GmbH). Aerosol \njet printing has been made using an IDS Nanojet Desktop system. Microelectrodes were printed \non 30mm Millicell culture plate inserts (PICM0RG50; Sigma Aldrich / Merck). The insert \nsubstrate is made of porous hydrophilic PTFE membrane featuring 0.4 μm pore sizes. For initial \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n15 \n \nbiocompatibility testing, 10 parallel lines of 10 mm length incrementally distanced by 1 mm were \nprinted mimicking MEA features. \nTo fabricate double side printed microelectrode schematics (Figure 1b,c), the membrane \nsubstrates were pierced using tungsten micro needles (<1µm tip diameter, 125 µm shaft diameter, \nWorld Precision Instruments). This enabled us to produce μm-sized pores that were filled by the \ninkjet printing with the conductive ink, providing a good conductivity pathway between the two \nsides of the membrane.  \nScanning electron microscopy (SEM) images were obtained with a scanning electron \nmicroscope (JEOL JCM-6000PLUS) operating at 15 kV . We used a physical vapor deposition \nsystem (Moorfield M307) to deposit a thin (approx. 100 nm) layer of gold on the studied samples, \nenabling SEM imaging.  \n \nOrganotypic culture. Cutting solution was prepared by dissolving the following components in \ndistilled water (500 mL): sucrose 30.75 g, KCl 0.095 g, MgSO4 1.23 g, NaH2PO4 0.0975g, \nNaHCO3 1.05 g, glucose 0.9 g, ascorbic acid 0.088g, taurine 0.0625 g, ethyl pyruvate 1.1 mL, \nCaCl2 0.0375 g. Artificial cerebrospinal fluid (aCSF) solution was prepared by mixing in distilled \nwater (500 mL): NaCl: 3.68 g, KCl 0.165 g, NaH2PO4 0.098 g, NaHCO3 1.05 g, glucose 0.99 g, \nMgCl2 0.062 g, CaCl2 0.147 g. Osmolarity of both solutions was in the range of 300-310 \nmOsm/L. Both solutions were sterilized by Stericup Quick Release Filter (Millipore; 0.22um PES \nMembrane) and carbogenated (95% O2/5% CO2) for at least 30 minutes before use. [3] \nInstruments and beakers were sterilized with autoclave (at 121ºC; Prestige Medical Classic \nMedia Autoclave), a microtome, and all the instruments were sprayed with 70% ethanol and \nallowed for full drying before use. The brains were extracted from rats (postnatal day 7; P7) \nhumanely culled via Animals (Scientific Procedures) Act 1986 (ASPA) Schedule 1 methods; \nethical approval was obtained prior the research and all procedures involving animals (rats) have \nbeen compliant with the Home Office (UK) guidelines / personal licenses (PP9211565) and the \nNC3R’s ARRIVE guidelines. The brain was sliced with a thickness of 350 µm in the ice-cold \ncutting solution. Freshly cut slices were transferred to aCSF continuously bubbled with 95% \nO2/5% CO2 and placed on a mesh to rest for 1 hour at room temperature. Afterwards, the slices \nwere transferred to the organotypic culture media allowing to flush out excess of aCSF. The \nculture media have been prepared using the following protocol: 50 mL Gibco™ Neurobasal™-A \nMedium was mixed with 1 mL Gibco™ B-27™ Supplement, 150 µL Gibco™ Gentamicin and \n125 µL GlutaMAX™ Supplement.  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n16 \n \nThe slices were placed on the 30mm Millicell culture plate inserts (PICM0RG50; Sigma \nAldrich / Merck) with and without microelectrodes (for reference). The inserts with \nmicroelectrodes were washed in an aqueous solution of PBS to remove excess PS1 and then \nsterilized by UV light (1 hour on each side). The inserts were maintained in wells with the culture \nmedia at 37ºC temperature and 5.0% CO2 supply of a culture incubator. The culture media was \nchanged for a fresh one once per 3-4 days to provide a proper nutrient supply. During the culture \nperiod (0-21 days), individual slices were removed from the wells and either fixed for histology or \nsubject to electrophysiological recordings. \nFixed slices were stained for histology by standard H&E (hematoxylin and eosin) staining \nkit (ab245880; datasheet and protocol - www.abcam.com/ab245880). Brightfield images were \ndigitized from a Toupcam eyepiece camera connected to a binocular microscope (GT Vision) at \nx40, and cell counts were performed using FIJI. \n \nElectrophysiological Recordings: Culture wells containing brain slices as prepared above were \ntransferred to one of two electrophysiology setups to test ensemble activity: \nGlass Microelectrode Rig consisted of an interface recording chamber (Scientific Systems \nDesign) filled with standard aCSF (see Organotypic Cultures above) and binocular dissecting \nmicroscope (Olympus) with dual electrodes connected to a local field potential amplifier (NPI \nElectronics EXT10-2F) and filter (NPI Electronics LHBF-48X). Glass microelectrodes (1-3MΩ) \nfilled with aCSF were placed upon recording pads creating a continuous connection ensuring that \nneither electrode nor pad was damaged. Ensemble activity was recorded at a total amplification of \n1000x, with low and high pass filters of 700 Hz and 0.5 Hz, respectively. Signals were digitized at \n2 kHz using a CED 1401 connected to a Windows PC running Spike2 version 8. \nSolid Electrode Rig was based on a Kerr Scientific In Vitro Brain Slice System, with the \nculture well placed in the central chamber and filled with aCSF. Insulated solid wire electrodes \n(Kerr Scientific) were placed onto the recording pads firmly, creating a slight deformation in the \nPTFE substrate to ensure maximal connection. Signals were amplified 250x with the Kerr isolated \namplifier, AC-coupled at 0.5Hz and low-pass filtered at 500Hz, digitized at 2kHz using a CED \n1401 connected to a Windows PC running Spike2 version 8. \nIn all cases, cultured slices were perfused with a minimal amount of aCSF bubbled with \n95% O2/5% CO2 and left in the chamber to equilibrate for 15 minutes. Locations of printed \nelectrode pads were determined under the microscope, and the pad(s) closest to the cell body layer \nof the hippocampus, or to layers 2/3 of the neocortex, were chosen. Multi-unit activity was \nelicited after a 10-minute baseline period via bath addition of 20 µM gabazine to disinhibit the \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n17 \n \nslices. Electrodes were removed from pads and placed into the aCSF after recording to ensure that \nmulti-unit activity was recorded via the contact pads rather than transmitted through the fluid. \n \n \nAcknowledgements \nP.L. and S.D.G. acknowledge support of the British Academy, Royal Academy of Engineering and \nRoyal Society (Academies Partnership in Supporting Excellence in Cross-disciplinary research \naward - APEX award, AA21\\100133 APEX Awards 2022). Dr. Emily Allwright and Dr. Neil \nChilton are acknowledged for technical assistance in aerosol jet printing using the IDS Nanojet \nsystem at Printed Electronics Ltd. \n \n \nData Availability Statement \nThe data that support the findings of this study are available from the corresponding author(s) \nupon reasonable request. \n \nReferences \n[1] Humpel, C., Organotypic Brain Slice Cultures: A Review. 2015, Neuroscience, 305, 86, \nhttps://doi.org/10.1016/j.neuroscience.2015.07.086 \n[2] Bak, A., Koch, H., van Loo, K. M. J., et al., Human Organotypic Brain Slice Cultures: A \nDetailed and Improved Protocol for Preparation and Long-Term Maintenance, 2024, J. \nNeurosci. Methods, 404, 110055, https://doi.org/10.1016/j.jneumeth.2023.110055 \n[3] Jones, R. S., da Silva, A. B., Whittaker, R. G., Woodhall, G. L., Cunningham, M. O., Human \nBrain Slices for Epilepsy Research: Pitfalls, Solutions and Future Challenges, 2016, J. \nNeurosci. 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Commun., 5, \n5259, https://doi.org/10.1038/ncomms6259  \n \n \n \nSupporting Information \nSupporting Information is available. \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint \n\n20 \n \n \n \nTable of Contents figure (110 mm × 20 mm) \n \n \n \n \n \nTable of Contents text: \nThe highly porous microelectrodes have been designed and printed on culture membranes \nallowing to record electrophysiological neural activity for rodent brain slices. To keep the \nbiocompatible nanoporous structure, the microelectrodes and insulative layer were fabricated on \nthe bottom of culture membranes with only small connector pads added on the top. \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 15, 2025. ; https://doi.org/10.1101/2025.05.12.653414doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}