The authors acknowledge funding from the European Union&#39;s Horizon 2020 research and innovation programme under grant agreement No. 882897-Search&#38;Rescue project and the PON project &#34;TEX-STYLE&#34; ARS01_00996, PNR 2015-2020.

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All efforts were made to reduce the number of animals for the project and to minimize their suffering.
1. Preparation of the developing solution, the etching solutions, the organic semiconductor solution, and the photolithographic masks
<ol>
	<li>Prepare the developing solution by diluting NaOH pellets in deionized water at a concentration of 175 mM. 
	NOTE: This is an exothermic reaction. If a plastic container is used, keep agitating the container until all pellets are completely dissolved.</li>
	<li>Prepare the titanium etching solution by diluting hydrofluoric acid (HF) in deionized water (1 part of concentrated 48% HF, 49 parts deionized water). 
	CAUTION: Hydrofluoric acid can easily penetrate the skin, causing severe damage to deep tissue layers. Rapid neutralization of HF is necessary to prevent tissue destruction, which may continue for days and result in severe injury or even death. The risks associated with HF are dependent on the concentration and the duration of contact with the acid. Use only under a fume hood using a face shield. Double gloving is also strongly recommended.</li>
	<li>Prepare the gold etching solution by mixing iodine, potassium iodide, and deionized water (for 250 g of solution, use 200 mL of deionized water, 20 g of KI, 5 g of I2). Stir the solution at room temperature for 1 h and leave it resting overnight before use.</li>
	<li>Prepare the semiconductor solution by dissolving 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS Pentacene) in anisole (1% in weight) and gently stirring for 2 h on a hot plate at 80 &#176;C. 
	NOTE: Keep agitating this solution. Use amber glass vials and/or store them in low light conditions.</li>
	<li>Prepare the desired photolithographic mask set with a vector graphics software. Prepare 5 masks for the entire process: the mask for the patterning of the floating gates (FGs); the mask for the opening of the vias and the sensing areas for the electrophysiological recordings; the mask for the self-alignment process; the mask for the patterning of the source, drain and control gate top contact; and the mask for the plasma activation of the pH channels. 
	&#8203;NOTE: Depending on the necessary resolution and the specific photolithographic setup, different kinds of masks could be used. In the case of the proposed devices (which have a maximum lateral resolution of 40 &#181;m), simple plastic flexible masks have been bought at a local photocopy shop.</li>
</ol>
2. Substrate selection and preparation
<ol>
	<li>Cut a 6 x 6 cm2 square of 250 &#181;m polyethylene terephthalate (PET) from a pristine PET sheet. 
	NOTE: Start with a slightly bigger substrate than the final device to have wide enough margins to allow for manipulation with standard lab tweezers without damaging it.</li>
	<li>Inspect the substrate with an optical microscope to exclude the presence of deep grooves and scratches. Carefully select the less scratched substrates as bigger imperfections may lead to the failure of the final device.</li>
	<li>Rinse the PET substrates with acetone, isopropyl alcohol, and deionized water (in this order) and dry them using streams of nitrogen. Store the substrates in clean plastic Petri dishes/containers.</li>
</ol>
3. FG: titanium deposition
<ol>
	<li>Pre-clean the substrates with plasma oxygen (30 s at 100 W) and place them on the substrate holder inside the vacuum chamber of the thermal evaporator.</li>
	<li>Place 60 mg of titanium in the crucible, close the shutter, and pump down the evaporation chamber until it reaches a vacuum level lower than 10-6 Torr. Increase the power of the evaporator until the crucible glows red and wait for 30 s. Open the shutter, increase the power to 60% (or until the crucible glows bright white), and wait for 60 s. Close the shutter and turn down the power.</li>
	<li>Remove the substrates from the evaporator; clean them using acetone, isopropyl alcohol, and deionized water; and dry them using streams of nitrogen. Perform a second oxygen plasma treatment (60 s at 200 W) to slightly oxidize the titanium surface.</li>
</ol>
4. FG patterning
<ol>
	<li>Place one substrate at a time on the spin coater placed inside a fume hood. Deposit 4 mL of photoresist on the substrate using a disposable plastic pipette. Use the following spin coating parameters to obtain a 2 &#181;m-thick photoresist layer: spin speed: 3000 rpm; spin time: 45 s; acceleration time: 0.5 s; deceleration time: 0.5 s.</li>
	<li>Soft bake the photoresist by placing the substrate on a hot plate (70 &#176;C for 5 min). Store the substrate inside an aluminum foil-wrapped Petri dish/plastic container to avoid direct light exposure. 
	NOTE: Avoid the suggested bake temperature (100 &#176;C for 50 s) to prevent substrate deformation. However, baking at a lower temperature for a longer time ensures good results.</li>
	<li>Place the device in a bromograph and position the plastic photolithographic mask with the desired FG layout onto the substrate. Expose to ultraviolet (UV) light from the top for 1 min, and carefully remove the mask, taking care to minimize the lateral movements of the mask over the substrate to avoid scratching it.</li>
	<li>Plunge the substrate for 5 s in a glass container filled with the developing solution (step 1.1). Quickly rinse it in deionized water and dry it under nitrogen. Use an optical microscope to look for underdeveloped/overdeveloped spots in the substrate. Repeat the immersion of the substrate in developing solution in case of underdevelopment.</li>
	<li>Etch the exposed titanium by submerging it in the titanium etching solution (step 1.2) for 15 s, rinse it with deionized water, and dry it using nitrogen. Optically inspect the substrate and remove the photoresist using acetone. Rinse the substrate with isopropyl alcohol and deionized water, and dry it with nitrogen.</li>
</ol>
5. Gate dielectric deposition
<ol>
	<li>Prepare the deposition chamber of the Parylene coater by distributing 2 mL of the adhesion promoter (silane - 3-(trimethoxysilyl)propyl methacrylate) on the deposition chamber walls using a laboratory wipe. Place 300 mg of Parylene C dimer (corresponding to a final thickness of 150 nm) onto the Parylene coater. Set the lower pressure value to 7 mbar and the higher pressure value to 10 mbar. After the deposition, clean the substrates with acetone, isopropyl alcohol, and deionized water, and dry them with nitrogen.</li>
</ol>
6. Opening of the sensing areas of the OCMFET for electrical activity recording and formation of the vias to access the back of the FGs
<ol>
	<li>Deposit the photoresist on the substrates using the same parameters of steps 4.1 and 4.2.</li>
	<li>Place the device in a bromograph and position the plastic photolithographic mask onto the substrate for the vias (circular openings with a diameter of 50 &#181;m over the sensing areas and 100 x 100 &#181;m2 openings over the FGs away from the sensing areas (referred to as back contact of the FGs in Figure 1 and Figure 2)) under a stereoscopic microscope to improve the alignment precision. Expose to UV light from the top for 1 min, and carefully remove the mask, taking care to minimize the lateral movements of the mask over the substrate to avoid scratching it. 
	NOTE: The vias on the side of the FGs away from the sensing area (shown as back contact of FGs in Figure 1 and Figure 2) are necessary for contact during the characterization of the transistor. Moreover, having electrical access to the FGs may be very useful for different types of functionalization (e.g., electrodeposition).</li>
	<li>Develop the photoresist as previously described in step 4.4. Expose the substrate with the patterned photoresist (which acts as a mask here) to oxygen plasma (180 s at 200 W) to remove the Parylene C from the sensing areas. 
	NOTE: The etching rate of Parylene C in an isotropic plasma cleaner at 200 W is approximately 90 nm/min. A slight over-etch is performed to further clean the sensing areas. The photoresist is also etched during the process. However, its thickness (2 &#181;m) is much higher than that of the Parylene C.</li>
	<li>Place the substrates into a glass container filled with acetone inside the ultrasonic bath for 10 s to remove the photoresist completely. Rinse the substrates with acetone, isopropyl alcohol, and water and dry them with nitrogen. 
	&#8203;NOTE: Using sonication instead of simply rinsing the substrates with acetone is crucial to prevent undesired folding and re-deposition of Parylene C fragments onto the surface of the sensing areas.</li>
</ol>
7. Self-alignment of source and drain with the FG
<ol>
	<li>Deposit the photoresist on the substrates using the same parameters of steps 4.1 and 4.2. Place the device in a bromograph and position onto the substrate a plastic photolithographic mask with simple black rectangles that completely cover the transistor&#39;s areas. Expose to UV light for 1 min from both the top and the bottom, and carefully remove the mask, taking care to minimize the lateral movements of the mask over the substrate to avoid scratching it. 
	&#8203;NOTE: With the double-sided exposure, the FGs act as photolithographic masks with respect to the bottom exposure, while the presence of the top mask ensures that only the photoresist present on the transistors&#39; channel remains unexposed.</li>
	<li>Develop the photoresist as previously described in step 4.4.</li>
</ol>
8. Gold deposition, channel formation, and patterning of the sources, drains, and control gates
<ol>
	<li>Clean the substrates with a gentle plasma treatment (30 s at 30 W) to promote the adhesion of the metal on the Parylene C and place them on the substrate holder inside the vacuum chamber of the thermal evaporator.</li>
	<li>Place 30 mg of gold in the crucible, close the shutter, and pump down the evaporation chamber until it reaches 10-5 Torr. Increase the power of the evaporator until the crucible glows red and wait for 30 s. Open the shutter, increase the power to 40% (or until the crucible glows bright white), wait for 60 s, close the shutter, and turn down the power.</li>
	<li>Place the substrates into an acetone container inside the ultrasonic bath for 10 s to lift off the photoresist, thus removing the gold from the transistors&#39; channel. Rinse the substrates with acetone, isopropyl alcohol, and water and dry them with nitrogen. Deposit the photoresist on the substrates using the same parameters of steps 4.1 and 4.2.</li>
	<li>Place the device in a bromograph and position onto the substrate a plastic photolithographic mask with the desired sources, drains, and control gate layout. Expose to UV light for 1 min from the top, and carefully remove the mask, taking care to minimize the lateral movements of the mask over the substrate to avoid scratching it.</li>
	<li>Develop the photoresist as described in step 4.4. Etch the exposed gold by submerging it in the gold etching solution (step 1.3) for 10 s, rinse it with deionized water, and dry it out using nitrogen. Optically inspect the substrate and remove the photoresist using acetone. Rinse with isopropyl alcohol and deionized water and dry it with nitrogen.</li>
</ol>
9. Deposition and activation of the Parylene C for pH sensing
<ol>
	<li>Deposit the photoresist on the substrates using the same parameters of steps 4.1 and 4.2.</li>
	<li>Place the device in a bromograph and position onto the substrate a plastic photolithographic mask with openings corresponding to the pH-sensing areas of the OCMFETs. Expose to UV light for 1 min from the top, and carefully remove the mask, taking care to minimize the lateral movements of the mask over the substrate to avoid scratching it.</li>
	<li>Develop the photoresist as described in step 4.4. Protect the whole device, except for the pH-sensing areas, with polyimide insulation tape (see the Table of Materials). Deposit a layer of 500 nm of Parylene C (corresponding to 1 g of Parylene C dimer) on the substrate using the same parameters described in step 5.1. 
	NOTE: The total Parylene C thickness on the pH-sensing areas is 650 nm. No silane is required for this deposition.</li>
	<li>Carefully remove the polyimide insulation tape. Expose the substrate to oxygen plasma (5 min and 30 s at 200 W) to activate the Parylene C on the pH-sensing areas of the OCMFETs. 
	NOTE: The polyimide insulation tape is necessary here to limit the Parylene C deposition. In fact, a simple lift-off using the photoresist does not give positive results due to the almost pinhole-free nature of the conformal coating obtained with Parylene C.</li>
	<li>Place the substrates into an acetone container inside the ultrasonic bath for 10 s to completely remove the photoresist. Rinse the substrates with acetone and isopropyl alcohol (no water) and dry them with nitrogen.</li>
</ol>
10. Semiconductor deposition, culturing chamber placing, and final cut-out of the device from the PET
<ol>
	<li>Place the substrates onto a hot plate at 50 &#176;C. Cast a droplet (1 &#181;L) of semiconductor solution (step 1.4) onto each channel area, cover the whole substrate with a lid, and let it dry out under a chemical hood for 30 min.</li>
	<li>Prepare the culturing chamber by printing an acrylonitrile butadiene styrene ring with an internal radius of 15 mm, a thickness of 1 mm, and a height of 7 mm with a 3D printer. Glue the culturing chamber onto the central part of the substrate using polydimethylsiloxane (ratio of the curing agent: 15% by weight). Cut out the device from the PET either manually or using a laser cutter.</li>
</ol>
11. Electrical characterization of transistors
<ol>
	<li>Characterize each transistor using a sourcemeter18,19,20,21 (see the Table of Materials). 
	NOTE: Both the output and the input characteristics should be measured to extrapolate the transistors&#39; parameters (mainly the carriers&#39; mobility, threshold voltage, ION/IOFF ratio, and subthreshold slope).</li>
</ol>

<ol>
	<li>Hubel, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tungsten+microelectrode+for+recording+from+single+units.">Tungsten microelectrode for recording from single units.</a> Science. 125 (3247), 549-550 (1957).</li><li>Verzeano, M., Negishi, K., Angeles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+in+cortical+and+thalamic+networks.+A+study+with+multiple+microelectrodes.">Neuronal activity in cortical and thalamic networks. A study with multiple microelectrodes.</a> Journal of General Physiology. 43 (6), 177-195 (1960).</li><li>Thomas, C. A., Springer, P. A., Loeb, G. E., Berwald-Netter, Y., Okun, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+miniature+microelectrode+array+to+monitor+the+bioelectric+activity+of+cultured+cells.">A miniature microelectrode array to monitor the bioelectric activity of cultured cells.</a> Experimental Cell Research. 74 (1), 61-66 (1972).</li><li>Grattarola, M., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+the+neuron-microtransducer+junction:+from+extracellular+to+patch+recording.">Modeling the neuron-microtransducer junction: from extracellular to patch recording.</a> IEEE Transactions on Biomedical Engineering. 40 (1), 35-41 (1993).</li><li>Wallace, K., Strickland, J. D., Valdivia, P., Mundy, W. R., Shafer, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiplexed+assay+for+determination+of+neurotoxicant+effects+on+spontaneous+network+activity+and+viability+from+microelectrode+arrays.">A multiplexed assay for determination of neurotoxicant effects on spontaneous network activity and viability from microelectrode arrays.</a> NeuroToxicology. 49, 79-85 (2015).</li><li>Bergveld, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+operation,+and+application+of+the+tool+for+electrophysiology.">Development, operation, and application of the tool for electrophysiology.</a> IEEE Transactions on Biomedical Engineering. 19 (5), 342-351 (1972).</li><li>Bergveld, P., Wiersma, J., Meertens, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+potential+recordings+by+means+of+a+field+effect+transistor+without+gate+metal,+called+OSFET.">Extracellular potential recordings by means of a field effect transistor without gate metal, called OSFET.</a> IEEE Transactions on Biomedical Engineering. 23 (2), 136-144 (1976).</li><li>Fromherz, P., Offenhausser, A., Vetter, T., Weis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neuron-silicon+junction:+a+Retzius+cell+of+the+leech+on+an+insulated-gate+field-effect+transistor.">A neuron-silicon junction: a Retzius cell of the leech on an insulated-gate field-effect transistor.</a> Science. 252 (5010), 1290-1293 (1991).</li><li>Martinoia, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+ISFET+array-based+microsystems+for+bioelectrochemical+measurements+of+cell+populations.">Development of ISFET array-based microsystems for bioelectrochemical measurements of cell populations.</a> Biosensors and Bioelectronics. 16 (9-12), 1043-1050 (2001).</li><li>Heer, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CMOS+microelectrode+array+for+the+monitoring+of+electrogenic+cells.">CMOS microelectrode array for the monitoring of electrogenic cells.</a> Biosensors and Bioelectronics. 20 (2), 358-366 (2004).</li><li>Berdondini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+pixel+sensor+array+for+high+spatio-temporal+resolution+electrophysiological+recordings+from+single+cell+to+large+scale+neuronal+networks.">Active pixel sensor array for high spatio-temporal resolution electrophysiological recordings from single cell to large scale neuronal networks.</a> Lab on a Chip. 9 (18), 2644-2651 (2009).</li><li>Maccione, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+functional+connectivity+estimation+on+low-density+neuronal+cultures+recorded+by+high-density+CMOS+micro+electrode+arrays.">Multiscale functional connectivity estimation on low-density neuronal cultures recorded by high-density CMOS micro electrode arrays.</a> Journal of Neuroscience Methods. 207 (2), 161-171 (2012).</li><li>Kibler, A. B., Jamieson, B. G., Durand, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+aspect+ratio+microelectrode+array+for+mapping+neural+activity+in+vitro.">A high aspect ratio microelectrode array for mapping neural activity in vitro.</a> Journal of Neuroscience Methods. 204 (2), 296-305 (2012).</li><li>Frega, M., Tedesco, M., Massobrio, P., Pesce, M., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+dynamics+of+3D+engineered+neuronal+cultures:+a+new+experimental+model+for+in-vitro+electrophysiology.">Network dynamics of 3D engineered neuronal cultures: a new experimental model for in-vitro electrophysiology.</a> Scientific Reports. 4, 5489 (2014).</li><li>Zuo, L., Yu, S., Briggs, C. A., Kantor, S., Pan, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+a+three-dimensional+multi-electrode+array+for+neuron+electrophysiology.">Design and fabrication of a three-dimensional multi-electrode array for neuron electrophysiology.</a> Journal of Biomechanical Engineering. 139 (12), (2017).</li><li>Spanu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+micro-electrode+array+for+in-vitro+neuronal+interfacing.">A three-dimensional micro-electrode array for in-vitro neuronal interfacing.</a> Journal of Neural Engineering. 17 (3), 036033 (2020).</li><li>Spanu, A., Martines, L., Bonfiglio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacing+cells+with+organic+transistors:+a+review+of+in+vitro+and+in+vivo+applications.">Interfacing cells with organic transistors: a review of in vitro and in vivo applications.</a> Lab on a Chip. 21 (5), 795-820 (2021).</li><li>Spanu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+organic+transistor-based+system+for+reference-less+electrophysiological+monitoring+of+excitable+cells.">An organic transistor-based system for reference-less electrophysiological monitoring of excitable cells.</a> Scientific Reports. 5, 8807 (2015).</li><li>Viola, F. A., Spanu, A., Ricci, P. C., Bonfiglio, A., Cosseddu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrathin,+flexible+and+multimodal+tactile+sensors+based+on+organic+field-effect+transistors.">Ultrathin, flexible and multimodal tactile sensors based on organic field-effect transistors.</a> Scientific Reports. 8, 8073 (2018).</li><li>Napoli, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electronic+detection+of+DNA+hybridization+by+coupling+organic+field-effect+transistor-based+sensors+and+hairpin-shaped+probes.">Electronic detection of DNA hybridization by coupling organic field-effect transistor-based sensors and hairpin-shaped probes.</a> Sensors. 18 (4), 990 (2018).</li><li>Spanu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reference-less+pH+sensor+based+on+an+organic+field+effect+transistor+with+tunable+sensitivity.">A reference-less pH sensor based on an organic field effect transistor with tunable sensitivity.</a> Organic Electronics. 48, 188-193 (2017).</li><li>Lundgaard, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+neuronal+glucose+uptake+heralds+activity-dependent+increases+in+cerebral+metabolism.">Direct neuronal glucose uptake heralds activity-dependent increases in cerebral metabolism.</a> Nature Communications. 6, 6807 (2015).</li><li>Zhang, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisensor-integrated+organs-on-chips+platform+for+automated+and+continual+in+situ+monitoring+of+organoid+behaviors.">Multisensor-integrated organs-on-chips platform for automated and continual in situ monitoring of organoid behaviors.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (12), 2293-2302 (2017).</li><li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+design+of+multifunctional+integrated+cell-based+biosensors+for+simultaneously+detecting+cell+acidification+and+extracellular+potential.">A novel design of multifunctional integrated cell-based biosensors for simultaneously detecting cell acidification and extracellular potential.</a> Biosensors and Bioelectronics. 24 (5), 1462-1468 (2009).</li><li>Maccione, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+algorithm+for+precise+identification+of+spikes+in+extracellularly+recorded+neuronal+signals.">A novel algorithm for precise identification of spikes in extracellularly recorded neuronal signals.</a> Journal of Neuroscience Methods. 177 (1), 241-249 (2009).</li><li>Spanu, A., Tedesco, M. T., Martines, L., Martinoia, S., Bonfiglio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+organic+neurophysiological+tool+for+neuronal+metabolic+activity+monitoring.">An organic neurophysiological tool for neuronal metabolic activity monitoring.</a> APL Bioengineering. 2 (4), 046105 (2018).</li><li>D&#237;az-Garc&#237;a, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+stimulation+triggers+neuronal+glycolysis+and+not+lactate+uptake.">Neuronal stimulation triggers neuronal glycolysis and not lactate uptake.</a> Cell Metabolism. 26 (2), 361-374 (2017).</li><li>Xie, Y., Dengler, K., Zacharias, E., Wilffert, B., Tegtmeier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+sodium+channel+blocker+tetrodotoxin+(TTX)+on+cellular+ion+homeostasis+in+rat+brain+subjected+to+complete+ischemia.">Effects of the sodium channel blocker tetrodotoxin (TTX) on cellular ion homeostasis in rat brain subjected to complete ischemia.</a> Brain Research. 652 (2), 216-224 (1994).</li><li>Caboni, A., Orgiu, E., Barbaro, M., Bonfiglio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+organic+thin-film+transistors+for+pH+monitoring.">Flexible organic thin-film transistors for pH monitoring.</a> IEEE Sensors Journal. 9 (12), 1963-1970 (2009).</li><li>Fraboni, B., Bonfiglio, A., Basiric&#242;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inkjet+printing+of+transparent,+flexible,+organic+transistors.">Inkjet printing of transparent, flexible, organic transistors.</a> Thin Solid Films. 520, 1291-1294 (2011).</li></ol>

The authors have no conflicts of interest to declare.

Unlike previous methods for the fabrication of OCMFETs for cellular applications18,29, the proposed method is specifically designed to prepare MOAs that can simultaneously detect electrical and metabolic cellular activity. Moreover, this approach to achieve pH sensitivity has the advantage of being compatible with standard fabrication protocols and does not involve any chemical modification of the sensing area (this aspect ensures the biocompatibility of the whole device). The pH sensitivity is achieved using the same material used as a gate dielectric (i.e., the biocompatible Parylene C), making this approach fast and reproducible.
The final result of this approach is a flexible, transparent, low-cost, and multisensing organic tool for in vitro cellular applications. The fact that this can be obtained using a single transistor structure and a simple physical modification of the sensing area adds to the advantages offered by the use of organic electronic materials and methods.&#160;Moreover, because the transduction principle of the OCMFET does not strictly depend on the specific semiconductor or FG material, the whole process can be modified and upscaled depending on the specific application.
A critical aspect of the proposed technique is related to the reproducibility of the plasma activation technique. To obtain consistent results, both the Parylene C thickness and its etching rate must be controlled. Frequent calibration of the Parylene C deposition process and the plasma cleaner are absolutely necessary. Other critical aspects, which also contribute to the reproducibility of the process, are the careful handling of the device and the deposition of the organic semiconductor. A simple drop-casting technique was used here, which intrinsically poses reproducibility limitations. To minimize those issues, as described in protocol step 10.1, the same amount of semiconductor solution should be used every time, and the solvent evaporation should be standardized as much as possible. Keeping a constant temperature using a hot plate and covering the substrate after each droplet deposition will help in slowing down the evaporation process. To further minimize this problem, the deposition technique (e.g., using an inkjet printing method) could be switched30.
A limitation of the proposed protocol stems from the nature of the functionalization of the OCMFET for pH sensing. The stability of the pH sensors is limited to a few weeks26. However, the stability window of the proposed approach is sufficiently large to cover the standard incubation times needed for neuronal culture growth (2-3 weeks). Other types of sensing area functionalization should be considered for longer experiments. The fabrication protocol uses a dedicated back contact, allowing electrical access to the FGs. This contact, which is left floating during the normal operation of the device, can be exploited for the electrical characterization of the device and the functionalization of the sensing areas using different techniques (e.g., electrodeposition).
This procedure represents a convenient way to prepare a multi-sensing device for cellular applications without the need for expansive materials or cleanroom facilities. Despite the performance and stability limitations due to the employment of an organic semiconductor and physical (not chemical) functionalization of the sensing area, similar approaches could be used to prepare low-cost (and potentially disposable), mechanically flexible, and optically transparent sensors and biosensors, which can provide researchers in cellular biology, tissue engineering, and neuroscience with novel specialized tools for studying cellular systems in vitro.

In vivo monitoring of electroactive cells, such as neurons and cardiomyocytes, represents a valid and powerful approach in fundamental research applications for the human brain, functional connectivity studies, pharmacology, and toxicology. The tools usually employed for such studies are mainly based on microelectrode arrays (MEAs)1,2,3,4,5 and increasingly more efficient and powerful field-effect devices (FEDs)6,7,8,9,10,11,12. These two families of devices allow the real-time monitoring and stimulation of the electrical activity of neurons and cardiomyocytes and are usually characterized by robustness, ease of use, and reliability. These features make MEAs and FEDs the gold standard for electrophysiological applications, being currently employed to interface with standard cellular cultures, organotypic brain slices, and tridimensional organoids13,14,15,16. Despite their widespread use and their impressive features, MEAs and FEDs present some limitations such as high cost, the rigidity of the materials, and the presence of a usually bulky reference electrode, which has to be placed in the measurement liquid environment and is necessary for proper operation of the devices.
To explore alternative solutions for cellular interfacing, much effort has been invested in the past decade into the study of electronic devices based on organic materials and innovative fabrication techniques17. Among the several organic devices studied to address the aforementioned limitations, a peculiar organic transistor called OCMFET has been recently proposed as a valid alternative to MEAs and FEDs18. In addition to the standard features offered by the organic electronics technology, such as low-cost materials and fabrication techniques, optimal mechanical and chemical properties, optical transparency, and biocompatibility, the OCMFET also offers an ultra-high charge sensitivity (due to its double-gated structure) without the need of an external reference electrode. Moreover, this organic sensor has the remarkable capability of sensing different analyte/physical parameters, depending on the specific functionalization of its sensing area, which is separated from the transistor area19,20. All these features can be conveniently exploited for the acquisition of different parameters within a cellular culture. In particular, in addition to being able to detect the neuronal/cardiac electrical activity, it is also possible to exploit the ultra-high pH sensitivity offered by the peculiar double-gated structure of the OCMFET by using a simple physical functionalization21 to reliably monitor the slight local pH variations caused by cellular metabolic activity.
In in vitro cell biosensing, the monitoring of cellular metabolic activity is a powerful indicator of the state of the culture and can be used to assess the cellular response to various stimuli, such as drug administration and electrical stimulation22,23. Moreover, in the specific case of neural applications, monitoring both the electrical and the metabolic activities is of great interest, particularly in pharmacology and toxicology24. With the intent of conveniently addressing the requirements of modern in vitro electrophysiology while at the same time offering all the advantages of the OCMFET, a device called Micro OCMFET Array (MOA) has been recently introduced. The MOA is an OCMFET-based array with specialized sensing areas specifically designed for in vitro cellular interfacing, enabling the multiparametric analysis of electrogenic cells cultures. In particular, two MOA channels have bigger sensing areas to maximize their sensitivity and can be selectively functionalized to monitor specific parameters of interest, such as the pH variations of the culture medium. The other OCMFETs in the structure act as extracellular electrical activity sensors. Figure 1 shows the structure of a 16 channel MOA. This capability, combined with the absence of an external reference electrode, makes the MOA a very interesting tool for in vitro applications. This work presents the step-by-step fabrication protocol of a multisensing MOA for the in vitro detection of the electrical and metabolic activities of neurons and cardiomyocytes. Figure 2 shows the main fabrication steps, the materials used, and the device structure.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-(Trimethoxysilyl)propyl methacrylate</td>
			<td>Sigma Aldrich</td>
			<td>440159</td>
			<td></td>
		</tr>
		<tr>
			<td>3D printer Makerbot Replicator 2x</td>
			<td>Makerbot</td>
			<td></td>
			<td>https://www.makerbot.gr/. Estimated price: 2k-3k euros.</td>
		</tr>
		<tr>
			<td>ABS filament</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anisole</td>
			<td>Sigma Aldrich</td>
			<td>296295</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromograph model Hellas</td>
			<td>Bungard</td>
			<td></td>
			<td>https://www.bungard.de/. Estimated price: 1k-2k euros.</td>
		</tr>
		<tr>
			<td>Gold</td>
			<td>Local seller</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrofluoric acid</td>
			<td>Sigma Aldrich</td>
			<td>695068</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Sigma Aldrich</td>
			<td>207772</td>
			<td></td>
		</tr>
		<tr>
			<td>Kapton tape</td>
			<td></td>
			<td></td>
			<td>polyimide insulation tape</td>
		</tr>
		<tr>
			<td>Laser cutter VLS2.30</td>
			<td>Universal Laser Systems</td>
			<td></td>
			<td>https://www.ulsinc.com/it. Estimated price: 20k euros.</td>
		</tr>
		<tr>
			<td>Multichannel Systems acquisition board</td>
			<td></td>
			<td></td>
			<td>www.multichannelsystems.com</td>
		</tr>
		<tr>
			<td>NaOH pellets</td>
			<td>Sigma Aldrich</td>
			<td>567530</td>
			<td></td>
		</tr>
		<tr>
			<td>Parylene C dimer</td>
			<td>SCS special coating systems coating</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS Silgard 184</td>
			<td>Sigma Aldrich</td>
			<td>761036</td>
			<td></td>
		</tr>
		<tr>
			<td>PDS 2010 LABCOATER 2 Parylene Deposition System</td>
			<td>SCS special coating systems</td>
			<td></td>
			<td>https://scscoatings.com/. Estimated price: 50k euros</td>
		</tr>
		<tr>
			<td>PET film biaxially oriented (thickness 0.25 mm)</td>
			<td>Goodfellow</td>
			<td>ES301450</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner Gambetti &#34;Tucano&#34;</td>
			<td>Gambetti</td>
			<td></td>
			<td>https://www.gambetti.it/. Estimated price: 20k euros.</td>
		</tr>
		<tr>
			<td>Positive photoresist AZ1518</td>
			<td>MicroChemicals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium iodide KI</td>
			<td>Sigma Aldrich</td>
			<td>221945</td>
			<td></td>
		</tr>
		<tr>
			<td>Source Meter 2636</td>
			<td>Keithley</td>
			<td></td>
			<td>https://it.farnell.com/. Estimated price: 18k euros</td>
		</tr>
		<tr>
			<td>Spin coater unit</td>
			<td>Ossila</td>
			<td></td>
			<td>https://www.ossila.com/. Estimated price: 2.5k euros.</td>
		</tr>
		<tr>
			<td>Stereoscopic microscope SMZ745T</td>
			<td>Nikon</td>
			<td></td>
			<td>https://www.microscope.healthcare.nikon. 
			com/. Estimated price: 2k-3k euros.</td>
		</tr>
		<tr>
			<td>Thermal evaporator unit</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TIPS pentacene (6,13-Bis(triisopropylsilylethynyl)-pentacene)</td>
			<td>Sigma Aldrich</td>
			<td>716006</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium wire</td>
			<td>Goodfellow</td>
			<td>TI005129</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>Falc Instruments</td>
			<td></td>
			<td>https://www.falcinstruments.it/. Estimated price: 1k euro.</td>
		</tr>
	</tbody>
</table>

in vitro multiparametric cellular analysis by micro organic charge-modulated field-effect transistor arrays

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in this field have driven technological progress in a back-and-forth process that ultimately determined the impressive achievements of the past 50 years. However, the most employed devices used for cellular interfacing (namely, the microelectrode arrays and microelectronic devices based on transistors) still present several limitations such as high cost, the rigidity of the materials, and the presence of an external reference electrode. To partially overcome these issues, there have been developments in a new scientific field called organic bioelectronics, resulting in advantages such as lower cost, more convenient materials, and innovative fabrication techniques.
Several interesting new organic devices have been proposed during the past decade to conveniently interface with cell cultures.&#160;This paper presents the protocol for the fabrication of devices for cellular interfacing based on the organic charge-modulated field-effect transistor (OCMFET). These devices, called micro OCMFET arrays (MOAs), combine the advantages of organic electronics and the peculiar features of the OCMFET to prepare transparent, flexible, and reference-less tools with which it is possible to monitor both the electrical and the metabolic activities of cardiomyocytes and neurons in vitro, thus allowing a multiparametric evaluation of electrogenic cell models.

The potential of the MOA has been validated here for both electrical activity recordings and metabolic activity monitoring. The precise estimation of the capabilities of the device to detect extracellular action potentials was based on a thorough characterization with rat cardiomyocytes cultures (particularly in primary rat cardiomyocytes measured at 8 days in vitro [DIV])18. Figure 3A shows a complete MOA with 16 OCMFETs. The top inset shows an example of a confluent rat cardiomyocyte culture adhering to the surface of the MOA. To highlight their health, the cells have been immunostained for the sarcomeric protein, tropomyosin, after the recording session. The bottom inset shows a single cardiomyocyte signal measured with an OCMFET.
Interestingly, the device could detect spontaneous electrical activity and the activity induced upon the administration of different chemicals, as shown in Figure 3B. This validation was crucial to demonstrate the feasibility of using this approach for electrogenic cell interfacing. Because of the array configuration, the MOA also allowed for the reconstruction of the propagation velocity of the cardiac signal, thus demonstrating the suitability of the system for the study of cellular networks (Figure 3C). For further validation to determine the actual detection limit of the device, the MOA was also tested with striatal neurons (21 DIV)18, with interesting results in terms of signal amplitude and the reliability of the recordings. As seen in Figure 3D, the OCMFET could amplify neuronal field potentials with remarkable stability, showing signal-to-noise ratios (SNRS) of up to 3.2 (in the same range as that of SNRs obtained with standard MEAs25). The recording setup consisted of custom multichannel electronics for the transistor biasing and the signal readout and conditioning. Each channel for the electrical recording has a first stage consisting of an I/V converter with a 1 M&#8486; feedback resistor and a 150 Hz-1.3 kHz bandpass filter with a voltage gain of 110. For all the presented measurements, the transistors were biased with VDS = VGS = -1 V. The A/D conversion and the data visualization and storing were performed using a data acquisition board (see the Table of Materials). All the measurement sessions were conducted inside a Faraday cage to minimize the electrical, environmental noise on the system.
As previously mentioned, by exploiting the simple physical functionalization presented in the protocol, it was possible to prepare highly sensitive pH sensors with a supernernstian response. Because of the presented fabrication approach, these pH devices could be integrated into an MOA and used to monitor the slight pH variations induced by the metabolic activity of primary hippocampal rat neurons26. In particular, as shown in Figure 4, only one of the two OCMFETs dedicated to low-frequency sensing was selectively functionalized to demonstrate the feasibility of the approach. This selective functionalization allowed the evaluation of the response of the two OCMFETs to chemically induced metabolic variations: in particular, a high metabolic state can be obtained using bicuculline (BIC), an inhibitor of GABA A receptors27, while a low metabolic state can be induced by the addition of tetrodotoxin (TTX), which eventually causes cellular death28. The recording setup consisted of the same custom multichannel electronics used for the electronic activity measurements.
Unlike the previous case, two dedicated channels were used to record the slow variations induced by the cellular metabolic activity. Each channel consisted of a simple circuit composed of two main blocks: an I/V converter with a 1 M&#8486; feedback resistor and a low-pass filter with a cut-off frequency of 10 Hz. The transistors were biased with VDS = VGS = -1 V, and all the measurements were carried out inside a Faraday cage to minimize the impact of external noise on the recordings (this is a particularly important aspect considering the low current fluctuations induced by the cellular metabolic activity). During the experiments, the cultures were maintained in a low-buffered culture medium, and the whole system was placed in a controlled environment (37 &#176;C and a continuous CO2/air flux). As expected, only the current of the pH-sensitive OCMFET could be modulated by the addition of 25 &#181;M BIC. This was further confirmed by the induction of the current variation by the corresponding variation of the cellular metabolic activity.
The same experiment was repeated after the addition of 10 &#181;M TTX, which resulted in a gradual slowing down of the cellular metabolism. Following the addition of the TTX, neither the pH-sensitive OCMFET nor the pH-insensitive one showed any response, thus demonstrating the efficacy of the approach. These results demonstrate the effectiveness of the proposed functionalization and its relative stability for up to 2 weeks. An important conclusion that can be drawn from the proposed experiments (both the electrical activity and the metabolic activity) is that it is possible to prepare different kinds of sensors by selectively functionalizing different OCMFETs within the same culturing area. This aspect represents a non-trivial achievement in biosensing for cellular applications because being able to monitor different parameters within the same cell culture is crucial for better characterization of the complexity of those biological systems.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62907/62907fig01.jpg" /> 
Figure 1: Top view of a 16 channel MOA for metabolic and electrical monitoring of electroactive cells. Scale bar = 1 cm. Abbreviations: OCMFETs = organic charge-modulated field-effect transistors; FG = floating gate; S/D = source/drain; MOA = micro OCMFET array. <a href="https://www.jove.com/files/ftp_upload/62907/62907fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62907/62907fig02.jpg" /> 
Figure 2: Main fabrication steps of an MOA for metabolic and electrical monitoring of electroactive cells. (A and B) The evaporated Ti film is patterned using a standard photolithographic process to prepare the floating gate of the OCMFETs. (C) Deposition of 15 nm of Parylene C. This layer, together with the native Ti oxide, acts as the gate dielectric of the transistors. (D and E) The Parylene C layer is patterned using plasma oxygen treatment. A patterned photoresist layer is used to selectively expose the sensing areas for the electrical recordings and the floating gate back contacts. (F) Patterning of the Au top contacts, namely the source, drain, control gate, and floating gate back contact. A self-alignment technique is used to improve the electrical performance of the device. (G-I) Deposition of the second layer of Parylene C on the sensing area of the OCMFETs for metabolic activity monitoring. After the oxygen plasma exposure, this layer will act as the pH-sensitive membrane (J). (K) Cross-section of a complete MOA (with materials) after the deposition of the organic semiconductor (TIPS Pentacene) and the culture chamber positioning. Abbreviations: OCMFETs = organic charge-modulated field-effect transistors; FG = floating gate; S/D = source/drain; MOA = micro OCMFET array; CG = control gate; PET = polyethylene terephthalate; Par C = Parylene C; TIPS = 6,13-bis(triisopropylsilylethynyl) pentacene; ABS = acrylonitrile butadiene styrene. <a href="https://www.jove.com/files/ftp_upload/62907/62907fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62907/62907fig03.jpg" /> 
Figure 3: Cellular electrical activity recordings with an MOA. (A) A confluent culture of rat cardiomyocytes (8 DIV) adhering to the surface of an MOA, fixed after a recording session and immunostained for the sarcomeric protein, tropomyosin (upper inset). Bottom inset: example of a single cardiomyocyte signal measured with an OCMFET. Scale bar = 150 &#181;m. (B) Chemical tuning of the electrical activity of a cardiomyocyte culture. The activity acceleration resulted from the addition of 100 mM norepinephrine, while suppression resulted from the addition of 100 mM verapamil. Left: beating frequency modulation; right: statistics on 5 OCMFETs-average and standard deviation: spike-count on 4 min of basal (129 &#177; 4.6), norepinephrine-mediated (280 &#177; 28.6) and verapamil-mediated activity (15 &#177; 1.9). (C) Reconstruction of the propagation of a cardiac signal. Right: raster plot of the spontaneous activity of the culture indicating the propagation of the signal from site 14 to site 41 (right). (D) Action potentials of striatal cells from rat embryo (21 DIV). This figure has been modified from 18. Abbreviations: OCMFET = organic charge-modulated field-effect transistor; MOA = micro OCMFET array; NE = norepinephrine; VER = verapamil; DIV = days in vitro. <a href="https://www.jove.com/files/ftp_upload/62907/62907fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62907/62907fig04.jpg" /> 
Figure 4: Metabolic activity recordings with an MOA. Response of the (A) pH-sensitive and (B) pH-insensitive channels of an MOA to the addition of 25 &#181;M BIC before and after the addition of 10 &#181;M of TTX. After the TTX addition, the behavior of the pH-sensitive channel becomes similar to that of the pH-insensitive one. In particular, no current variation can be observed after the BIC addition due to the TTX-induced cellular death. (C) MOA for metabolic activity recordings. The pH-sensitive and the pH-insensitive OCMFETs are outlined in green and red, respectively. Inset: healthy hippocampal neurons cultured onto the device after 15 DIV. Scale bar = 50 &#181;m. This figure has been modified from 26. Abbreviations: OCMFET = organic charge-modulated field-effect transistor; MOA = micro OCMFET array; BIC = bicuculline; TTX = tetrodotoxin; DIV = days in vitro. <a href="https://www.jove.com/files/ftp_upload/62907/62907fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays at JoVE.com

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Here, we present the fabrication protocol of an organic charge-modulated field-effect transistor (OCMFET)-based device for in vitro cellular interfacing. The device, called a micro OCMFET array, is a flexible, low-cost, and reference-less device, which will enable the monitoring of the electrical and metabolic activities of electroactive cell cultures.

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in ...

in-vitro-multiparametric-cellular-analysis-micro-organic-charge

Research

JoVE Journal

Bioengineering

2.3K Views.  University of Cagliari. Here, we present the fabrication protocol of an organic charge-modulated field-effect transistor (OCMFET)-based device for in vitro cellular interfacing. The device, called a micro OCMFET array, is a flexible, low-cost, and reference-less device, which will enable the monitoring of the electrical and metabolic activities of electroactive cell cultures.

Video: In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.