Nanoscale geometry determines mechanical biocompatibility of vertically aligned nanofibers

Vertically aligned carbon nanofibers (VACNFs) are promising material candidates for neural biosensors due to their ability to detect neurotransmitters in physiological concentrations. However, the expected high rigidity of CNFs could induce mechanical mismatch with the brain tissue, eliciting formation of a glial scar around the electrode and thus loss of functionality. We have evaluated mechanical biocompatibility of VACNFs by growing nickel-catalyzed carbon nanofibers of different lengths and inter-fiber distances. Long nanofibers with large inter-fiber distance prevented maturation of focal adhesions, thus constraining cells from obtaining a highly spread morphology that is observed when astrocytes are being contacted with stiff materials commonly used in neural implants. A silicon nanopillar array with 500 nm inter-pillar distance was used to reveal that this inhibition of focal adhesion maturation occurs due to the surface nanoscale geometry, more precisely the inter-fiber distance. Live cell atomic force microscopy was used to confirm astrocytes being significantly softer on the long Ni-CNFs compared to other surfaces, including a soft gelatin hydrogel. We also observed hippocampal neurons to mature and form synaptic contacts when being cultured on both long and short carbon nanofibers, without having to use any adhesive proteins or a glial monoculture, indicating high cytocompatibility of the material also with neuronal population. In contrast, neurons cultured on a planar tetrahedral amorphous carbon sample showed immature neurites and indications of early-stage apoptosis. Our results demonstrate that mechanical biocompatibility of biomaterials is greatly affected by their nanoscale surface geometry, which provides means for controlling how the materials and their mechanical properties are perceived by the cells.Peer reviewe

Nanoscale geometry determines mechanical biocompatibility of vertically aligned nanofibers

Vertically aligned carbon nanofibers (VACNFs) are promising material candidates for neural biosensors due to their ability to detect neurotransmitters in physiological concentrations. However, the expected high rigidity of CNFs could induce mechanical mismatch with the brain tissue, eliciting formation of a glial scar around the electrode and thus loss of functionality. We have evaluated mechanical biocompatibility of VACNFs by growing nickel-catalyzed carbon nanofibers of different lengths and inter-fiber distances. Long nanofibers with large inter-fiber distance prevented maturation of focal adhesions, thus constraining cells from obtaining a highly spread morphology that is observed when astrocytes are being contacted with stiff materials commonly used in neural implants. A silicon nanopillar array with 500 nm inter-pillar distance was used to reveal that this inhibition of focal adhesion maturation occurs due to the surface nanoscale geometry, more precisely the inter-fiber distance. Live cell atomic force microscopy was used to confirm astrocytes being significantly softer on the long Ni-CNFs compared to other surfaces, including a soft gelatin hydrogel. We also observed hippocampal neurons to mature and form synaptic contacts when being cultured on both long and short carbon nanofibers, without having to use any adhesive proteins or a glial monoculture, indicating high cytocompatibility of the material also with neuronal population. In contrast, neurons cultured on a planar tetrahedral amorphous carbon sample showed immature neurites and indications of early-stage apoptosis. Our results demonstrate that mechanical biocompatibility of biomaterials is greatly affected by their nanoscale surface geometry, which provides means for controlling how the materials and their mechanical properties are perceived by the cells.</p

Substraatin mekaanisten ominaisuuksien vaikutus aivosolujen solumekaniikkaan

While an animal body contains a wide range of tissue types, most cells are subjected only to soft environments in vivo, neural cells in particular. Living cells are however able to sense mechanical properties of their surroundings, which plays an essential role in regulating cell behavior. Acknowledging this mechanosensory nature of cells is becoming ever more important in making accurate cell/organ models or treating various pathologies with implants. While soft materials are readily available, they usually lack the functionalities necessary in sensing electrical or chemical signals from the cells. Thus, mechanical biocompatibility of the typically used functional materials must be assessed. Atomic force microscopy was used in this study to measure elasticity of living cells on various materials with different mechanical properties, including vertically aligned carbon nanofibers. Two neural cell lines, PC-12 Adh and C6, were found to show similar level of cytoskeletal strengthening on both soft hydrogel and carbon nanofibers, indicating both samples appearing soft from the cellular perspective. Intriguingly, carbon nanofibers are also known to be both electrically and electrochemically active functional materials. The results obtained here provide an early indication for mechanical biocompatibility of vertically aligned carbon nanofibers, further supporting their use as a sensory material for measuring the behavior of neural cells.Eläinsolut elävät kudoksissa, jotka ovat pääsääntöisesti pehmeitä materiaaleja muutamaa poikkeusta lukuun ottamatta. Koska solut kykenevät aistimaan ympäristönsä mekaanisia ominaisuuksia, voivat ne myös muuntaa käyttäytymistään ympäristönsä mekaanisten signaaleiden seurauksena. Tämän on havaittu olevan keskeisessä roolissa monissa solun toimintaa säätelevissä tapahtumissa, minkä seurauksena ympäristön mekaaniset ominaisuudet tulee ottaa huomioon tehtäessä entistä tarkempia solumalleja kuin myös suunnitellessa pitkäaikaisia implantteja. Pinnan kovuuden vaikutusta solujen sisäiseen mekaniikkaan mitattiin tässä työssä atomivoimamikroskopialla ja fluoresenssikuvantamisella. Hermostosta peräisin olevien PC-12 Adh ja C6 -solulinjojen välillä havaittiin samankaltaista käyttäytymistä: solut näyttäytyivät yhtä kovina sekä vertikaalisesti kasvatettujen hiilinanokuitujen että pehmeän hydrogeelin päällä, mikä indikoi solujen kokevan nämä materiaalit yhtä pehmeinä. Hiilinanoputket voivat toimia sekä normaalina sähköisenä elektrodina että sähkökemiallisena elektrodina. Koska hermoston solujen ympäristö on luontaisesti erittäin pehmeää, viittaa tämä löydös parempaan mekaaniseen yhteensopivuuteen vertikaalisten hiilinanoputkien päällä verrattuna normaalisti käytettyihin elektrodimateriaaleihin, jotka ovat varsin kovia

Ultraherkät ja selektiiviset anturit hermovälittäjäaineiden reaaliaikaiseen mittaamiseen aivosiruteknologiassa

Neurological diseases account annually up to 10 million deaths and the number is expected to increase as the population ages. Up to date, drug discovery has had 0% success rate to find disease modifying treatment for central nervous system diseases, mainly arising from extremely poor translation of results from animal studies to us humans. To overcome this limitation, human cellbased brain models are being rapidly adopted into academic neuroscientific research but also in the pharmaceutical industry. Unfortunately, the use of these brain models is limited by lack of highthroughput characterization methods. Electrochemical measurements can be used to examine the condition of specific cell types in the brain models, however this is severely complicated by the highly-fouling nature of cell culture medium. As complex in vitro brain models are extremely delicate, electrochemical recordings must be performed in the culture medium instead of highly clean electrolytes. By using electrochemical sensors prepared from single-walled carbon nanotube (SWCNT) films, real-time detection of neurotransmitters at nanomolar sensitivity was demonstrated in culture medium for the first time. Arising from the sensitivity of brain model cultures, biocompatibility of the sensor materials is essential because otherwise the brain-on-a-chip will lack the "brain" component. Healthy development of all the most-sensitive and complex in vitro brain models were observed with the SWCNT electrodes, including primary dopaminergic neurons, induced human pluripotent stem cell-derived neurons, but also human brain organoids. Lastly, many biological characterization techniques are based on inverse microscopy and thus, optical transparency is necessary. While transparent substrates could be used to obtain partial visibility for non-transparent microelectrode array, the electrode areas and electrical wiring remain out of sight. Contrasting that, the SWCNT films are highly conductive and can be made with 90% transparency to obtain complete visibility. While many materials are claimed to meet the requirements of brain-on-a-chip devices, more thorough investigation reveals that such claims are mistakenly made and actually only concern individual components from the ensemble of strict specifications. Furthermore, these earlier claims have been made based on highly simplified and misleading experimental settings. SWCNTs are thus a very unique electrode material, as they are currently the only material fulfilling all the requirements of brain-on-a-chip devices, paving way for broader adoption of the technology.Neurologiset sairaudet ovat vuosittain osallisena jopa 10 miljoonassa kuolemassa ja määrän odotetaan kasvavan ikääntyvän väestön lisääntyessä. Toistaiseksi saatavilla on ainoastaan oireita lievittäviä lääkkeitä, eikä sairauksia parantavia kohdennettuja lääkkeitä ole pystytty kehittämään. Vaikka eläinkokeissa onkin saatu lupaavia tuloksia kohdennettujen lääkkeiden käytöstä neurologisten sairauksien hoidossa, niiden tuloksia ei ole pystytty toistamaan ihmisissä. Haasteet kokeiden toistamisessa selittyvät muun muassa eroilla ihmisen ja koe-eläimen genomissa sekä hermostossa itsessään, minkä vuoksi tauti- ja lääketutkimuksessa olisi optimaalisinta käyttää ihmisen hermoston soluja. Aivo-organoidien ja muidenkin aivomallien käyttöön lääketutkimuksessa liittyy kuitenkin monia vaikeuksia, joista merkittävin kytkeytyy organoidien karakterisoimisen hitauteen. Mikäli organoidien vastetta eri lääkeaineille ei voida karakterisoida riittävän nopeasti tai monipuolisesti, lääkkeiden kehitys hidastuu merkittävästi. Sähkökemiallisia mittauksia voidaan hyödyntää organoidien olotilan tutkimisessa, erityisesti kun tavoitteena on tutkia tiettyä hermosolutyyppiä kaikkien solutyyppien joukosta. Sähkökemiallisia mittauksia kuitenkin vaikeuttaa elatusaineiden aiheuttama elektrodien likaantuminen, joka pienentää sensoreiden herkkyyttä merkittävästi. Tässä väitöskirjassa kehitettiin yksiseinämäisistä hiilinanoputkista valmistettuja sähkökemiallisia antureita hermovälittäjäaineiden reaaliaikaiseen mittaamiseen monimutkaisista aivo-malleista. Koska ihmisaivoja mahdollisimman tarkasti mallintavat aivomallit ovat erittäin herkkiä ympäristön vaikutuksille, sensorimateriaalien täytyy olla äärimmäisen bioyhteensopivia. Muutoin soluviljelmät muuttuvat epänormaaleiksi tai äärimmäisissä tapauksissa jopa tuhoutuvat kokonaan. Tässä väitöskirjassa havaittiin, että yksiseinämäisistä hiilinanoputkista tehdyt elektrodit mahdollistivat myös kaikkein herkimpienkin in vitro aivomallien selviytymisen elektrodin päällä. Biologisissa koejärjestelyissä käytetyistä karakterisointitekniikoista suurin osa perustuu käänteiseen mikroskopiaan, eli objektiivi sijaitsee näytteen alapuolella. Tämän vuoksi myös elektrodin olisi oltava läpinäkyvä, jotta sen päällä olevia aivomalleja voitaisiin tutkia mikroskopian menetelmin. Sähkökemiallisissa antureissa käytetyistä materiaaleista vain murto-osa on läpinäkyviä. Yksiseinämäisistä hiilinanoputkista voidaan kuitenkin valmistaa sähköisesti johtavia kalvoja yli 90 % läpinäkyvyydellä, kuten tässä väitöskirjassa on osoitettu. Yksiseinämäisistä hiilinanoputkista tehdyt ohutkalvot ovatkin siten hyvin ainutlaatuinen elektrodimateriaali, täyttäen kaikki aivosiru-teknologian vaatimukset

Ascorbic acid does not necessarily interfere with the electrochemical detection of dopamine

Funding Information: We wish to thank MSc Touko Liljeström for kindly providing us the ta-C samples and Canatu Oy (Finland) for providing the SWCNT electrode material required for this study. This work was supported by European Union’s Horizon 2020 research and innovation programme H2020-FETPROACT-2018-01 (No. 824070). Publisher Copyright: © 2022, The Author(s). | openaire: EC/H2020/824070/EU//CONNECTIt is widely stated that ascorbic acid (AA) interferes with the electrochemical detection of neurotransmitters, especially dopamine, because of their overlapping oxidation potentials on typical electrode materials. As the concentration of AA is several orders of magnitude higher than the concentration of neurotransmitters, detection of neurotransmitters is difficult in the presence of AA and requires either highly stable AA concentration or highly selective neurotransmitter sensors. In contrast to the common opinion, we show that AA does not always interfere electrochemical detection of neurotransmitters. The decay of AA is rapid in cell culture medium, having a half-time of 2.1 hours, according to which the concentration decreases by 93% in 8 hours and by 99.75% in 18 hours. Thus, AA is eventually no longer detected by electrodes and the concentration of neurotransmitters can be effectively monitored. To validate this claim, we used unmodified single-wall carbon nanotube electrode to measure dopamine at physiologically relevant concentration range (25–1000 nM) from human midbrain organoid medium with highly linear response. Finally, AA is known to affect dopamine oxidation current through regeneration of dopamine, which complicates precise detection of small amounts of dopamine. By designing experiments as described here, this complication can be completely eliminated.Peer reviewe

Real-time selective detection of dopamine and serotonin at nanomolar concentration from complex in vitro systems

Funding Information: We wish to thank Canatu Oy, Finland, for kindly providing us the SWCNT network samples, and PhD Toni Pasanen for kindly providing us the antireflective black silicon material that was used in upright fluorescence imaging. We acknowledge the provision of facilities by Aalto University at OtaNano - Micronova Nanofabrication Centre and we would also like to thank the Biomedicum Imaging Unit (BIU), Helsinki, for microscopy services. This work was supported by European Union's Horizon 2020 research and innovation programme H2020-FETPROACT-2018-01 (No. 824070), Doctoral Progamme in Drug Research (University of Helsinki), and Sigrid Juselius Foundation. Funding Information: We wish to thank Canatu Oy, Finland, for kindly providing us the SWCNT network samples, and PhD Toni Pasanen for kindly providing us the antireflective black silicon material that was used in upright fluorescence imaging. We acknowledge the provision of facilities by Aalto University at OtaNano - Micronova Nanofabrication Centre and we would also like to thank the Biomedicum Imaging Unit (BIU), Helsinki, for microscopy services. This work was supported by European Union's Horizon 2020 research and innovation programme H2020-FETPROACT-2018-01 (No. 824070 ), Doctoral Progamme in Drug Research (University of Helsinki) , and Sigrid Juselius Foundation . Publisher Copyright: © 2023 The Authors | openaire: EC/H2020/824070/EU//CONNECTElectrochemical sensors provide means for real-time monitoring of neurotransmitter release events, which is a relatively easy process in simple electrolytes. However, this does not apply to in vitro environments. In cell culture media, competitively adsorbing molecules are present at concentrations up to 350 000-fold excess compared to the neurotransmitter-of-interest. Because detection of dopamine and serotonin requires direct adsorption of the analyte to electrode surface, a significant loss of sensitivity occurs when recording is performed in the in vitro environment. Despite these challenges, our single-walled carbon nanotube (SWCNT) sensor was capable of selectively measuring dopamine and serotonin from cell culture medium at nanomolar concentration in real-time. A primary midbrain culture was used to prove excellent biocompatibility of our SWCNT electrodes, which is a necessity for brain-on-a-chip models. Most importantly, our sensor was able to electrochemically record spontaneous transient activity from dopaminergic cell culture without altering the culture conditions, which has not been possible earlier. Drug discovery and development requires high-throughput screening of in vitro models, being hindered by the challenges in non-invasive characterization of complex neuronal models such as organoids. Our neurotransmitter sensors could be used for real-time monitoring of complex neuronal models, providing an alternative tool for their characterization non-invasively.Peer reviewe

Nanoscale engineering to control mass transfer on carbon-based electrodes

Publisher Copyright: © 2022 The Author(s)Here we use an electrode consisting of carbon nanofibers (CNFs), the lengths and surface population density of which can be effectively controlled. It is shown that (i) a thin liquid layer forms when the thickness of the diffusion layer has a specific ratio to the dimensions of the nanostructured carbon surface. (ii) This leads to a decrease in the peak potential difference and a subsequent increase in the apparent heterogeneous electron transfer (HET) constant, both of which could be interpreted as a result of increased catalytic activity. (iii) However, we show that this explanation is not likely, as our materials are chemically identical, and we use an outer sphere redox (OSR) probe to minimize any specific chemical interactions. On the contrary, (iv) the results clearly show that the observed behavior is caused by a combination of the formation of a thin liquid layer and the increased apparent surface area of the electrodes.Peer reviewe

Shape Memory Polymer-Based Insertable Electrode Array towards Minimally Invasive Subdural Implantation

Publisher Copyright: CCBY Copyright: Copyright 2021 Elsevier B.V., All rights reserved. | openaire: EC/H2020/824070/EU//CONNECTMinimally invasive implantation of subdural electrodes can dramatically benefit the patients with various neurological diseases. In modern clinical practice, the implantation procedure of the electrode arrays remains traumatic for patients and increases postoperative infection risk. Here we report a design and insertion technique of thermally activated shape-memory polymer-based electrode array that can recover up to ten times length deformation. The compressed four-centimeter wide array can be easily packed into a three-millimeter diameter tube and subsequently deployed thought five-millimeter opening in a restricted space between a brain phantom and a simulated skull. The mechanical properties of the developed array are comparable to the materials traditionally employed for the purpose, and the electrical and signal recording properties are preserved after shape deformation and recovery. Additionally, the array is biocompatible and exhibits conformability to a curvy brain surface. The results demonstrate that insertion of the electrode array through a small hole into a restricted space similar to subdural cavity is possible, which may inspire future solution of minimal invasive implantation for patients suffering from epilepsy, amyotrophic lateral sclerosis or tetraplegia.Peer reviewe