JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Bioengineering

Fokuseret Ionstrålithografi til etch Nano-arkitekturer i Mikroelektroderne

Published: January 19, 2020
doi:
10.3791/60004
, , , ,
1Department of Electrical Engineering and Computer Science,University of Michigan, 2Veteran Affairs Ann Arbor Healthcare System, 3Department of Neurology, School of Medicine,University of Michigan, 4Department of Biomedical Engineering,University of Michigan, 5Michigan Center for Materials Characterization,University of Michigan, 6Carl Zeiss SMT, Inc.

Summary

Vi har vist, at ætsning af Nano-arkitektur i intracortical mikroelektrode enheder kan reducere den inflammatoriske respons og har potentiale til at forbedre elektrofysiologiske optagelser. Metoderne beskrevet heri skitserer en tilgang til etch Nano-arkitekturer i overfladen af ikke-funktionelle og funktionelle enkelt skaft silicium intracortical mikroelektroder.

Abstract

Med fremskridt inden for elektronik og fabrikation teknologi, har intracortical mikroelektroderne gennemgået betydelige forbedringer gør det muligt at fremstilling af sofistikerede mikroelektroderne med større opløsning og udvidede kapaciteter. Fremskridtene inden for fabrikation teknologi har støttet udviklingen af biomimetiske elektroder, som har til formål at gnidningsløst integreres i hjernen parenchyma, reducere den neuroinflammatoriske respons observeret efter elektrode indsættelse og forbedre kvaliteten og levetiden for elektrofysiologiske optagelser. Her beskriver vi en protokol til at ansætte en biomimetisk tilgang for nylig klassificeret som nano-arkitektur. Brugen af fokuseret ion Beam litografi (FIB) blev udnyttet i denne protokol til etch specifikke Nano-arkitektur funktioner i overfladen af ikke-funktionelle og funktionelle enkelt skaft intracortical mikroelektroder. Ætsning af nanoarkitekturer i elektrodeoverfladen indikerede mulige forbedringer af biokompatibiliteten og funktionaliteten af den implanterede anordning. En af fordelene ved at bruge FIB er evnen til at etch på fremstillede enheder, i modsætning til under fabrikation af enheden, lette grænseløs muligheder for at ændre talrige medicinske enheder efter fremstilling. Den protokol, der præsenteres heri, kan optimeres til forskellige materialetyper, nanoarkitektur funktioner og typer af enheder. Augmenting overfladen af implanterede medicinsk udstyr kan forbedre enhedens ydeevne og integration i vævet.

Introduction

Intracortical mikroelektroder (IME) er invasive elektroderne, som giver et middel til direkte grænseflade mellem eksterne enheder og neuronal populationer inde i hjernebarken1,2. Denne teknologi er et uvurderligt værktøj til optagelse af neurale aktionspotentialer for at forbedre forskernes evne til at udforske neuronal funktion, fremme forståelsen af neurologiske sygdomme og udvikle potentielle terapier. Intracortical mikroelektrode, der anvendes som en del af hjernen Machine Interface (BMI) systemer, muliggør registrering af handlings potentialer fra en enkelt eller små grupper af neuroner til at detektere motoriske hensigter, der kan bruges til at producere funktionelle udgange3. Faktisk har BMI-systemer med succes været anvendt til proteser og terapeutiske formål, såsom erhvervet sensorisk rytmekontrol til at betjene en computer markør hos patienter med Amyotrofisk lateral sklerose (ALS)4 og rygmarvsskader5 og genoprette bevægelsen i mennesker, der lider af kronisk tetraplegi6.

Desværre, IES ofte undlader at registrere konsekvent over tid på grund af flere svigt tilstande, der omfatter mekaniske, biologiske og materielle faktorer7,8. Den neuroinflammatoriske respons, der opstår efter elektrodens implantation, menes at være en betydelig udfordring, der bidrager til elektrode svigt9,10,11,12,13,14. Den neuroinflammatoriske respons initieres under den indledende indsættelse af IME, som severs blod-hjerne barrieren, skader den lokale hjerne parenkym og forstyrrer gliaceller og neuronal netværk15,16. Dette akutte respons er karakteriseret ved aktivering af gliacceller (mikroglia/makrofager og astrocytter), som frigiver pro-inflammatoriske og neurotoksiske molekyler omkring implantationsstedet17,18,19,20. Den kroniske aktivering af gliaceller celler resulterer i en fremmedlegeme reaktion karakteriseret ved dannelsen af et gliaceller ar isolerer elektroden fra sundt hjernevæv7,9,12,13,17,21,22. I sidste ende, hindrer elektrodens evne til at registrere neuronal handling potentialer, på grund af den fysiske barriere mellem elektroden og neuroner og degeneration og død af neuroner23,24,25.

Den tidlige fiasko af intracortical mikroelektroder har medført betydelig forskning i udviklingen af næste generation af elektroder, med vægt på biomimetiske strategier26,27,28,29,30. Af særlig interesse for den protokol, der er beskrevet her, er brugen af Nano-arkitektur som en klasse af biomimetiske overflade ændringer for IME’er31. Det er blevet fastslået, at overflader, der efterligner arkitekturen i det naturlige in vivo-miljø, har et forbedret biokompatibelt respons32,33,34,35,36. Således, hypotesen overbevisende denne protokol er, at diskontinuitet mellem den rå arkitektur af hjernevæv og glat arkitektur af de intracortical mikroelektroder kan bidrage til den neuroinflammatoriske og kroniske fremmedlegeme respons på implanterede i’er (for en fuld anmeldelse henvise til Kim et al.31). Vi har tidligere vist, at udnyttelsen af Nano-arkitektur funktioner svarende til hjernens ekstracellulære matrix arkitektur reducerer astrocyt inflammatoriske markører fra celler dyrket på nano-arkitekturerede substrater, sammenlignet med flade kontrol overflader i både in vitro og ex vivo modeller af neuroinflammation37,38. Desuden har vi vist anvendelsen af fokuseret ion Beam (FIB) litografi til etch Nano-arkitekturer direkte på silicium sonder resulterede i signifikant øget neuronal levedygtighed og lavere ekspression af pro-inflammatoriske gener fra dyr implanteret med nano-arkitektur sonder sammenlignet med den glatte kontrolgruppe26. Derfor er formålet med den protokol, der præsenteres her, at beskrive brugen af FIB litografi til etch Nano-arkitekturer på fremstillede intracortical mikroelektrode anordninger. Denne protokol er designet til at etch Nano-arkitektur mellemstore funktioner i silicium overflader af intracortical mikroelektrode skafter udnytte både automatiserede og manuelle processer. Disse metoder er ukomplicerede, reproducerbare, og kan helt sikkert optimeres til forskellige enheds materialer og ønskede funktions størrelser.

Protocol

Bemærk: gør følgende trin, mens du bærer det rigtige personlige værnemidler, såsom en laboratorie frakke og handsker. 1. montering af ikke-funktionel silicium sonde til fokuseret ionstråle (FIB) litografi Bemærk: for den komplette procedure, der beskriver fremstillingen af SOI wafer med 1.000-sonder, henvises der til Ereifej et al.39. Isoler en strimmel af 2-3 silicium sonder fra silicium på isolator (SOI) wafer indeholdend…

Representative Results

FIB ætset Nano-arkitektur på overfladen af enkelt skaft intracortical probesVed hjælp af de beskrevne metoder blev intracortical sonder ætset med specifikke nanoarkitekturer efter etablerede protokoller39. Dimensioner og form af Nano-arkitektur design, der er beskrevet i disse metoder blev implementeret fra tidligere in vitro-resultater, der skildrer et fald i gliaceller celle reaktivitet, når kulturperler med nano-arkitektur design beskrevet her37…

Discussion

Den fabrikations protokol skitseret her udnytter fokuseret ion Beam litografi til effektivt og reproducerbart etch Nano-arkitekturer i overfladen af ikke-funktionelle og funktionelle enkelt skaft silicium mikroelektroer. Fokuseret ionstråle (FIB) litografi giver mulighed for selektiv ablation af substrat overfladen ved hjælp af en fint fokuseret ionstråle50,51. FIB er en Direct-Write teknik, der kan producere forskellige funktioner med nanoskala opløsning og …

Disclosures

The authors have nothing to disclose.

Acknowledgements

Denne undersøgelse blev støttet af USA (USA) Department of Veterans Affairs rehabilitering forskning og udvikling service Awards: #RX001664-01A1 (CDA-1, Ereifej) og #RX002628-01A1 (CDA-2, Ereifej). Indholdet repræsenterer ikke de synspunkter, som det amerikanske Department of Veterans Affairs eller den amerikanske regering. Forfatterne vil gerne takke FEI Co. (nu en del af Thermo Fisher Scientific) for personale assistance og brug af instrumentering, som hjalp med at udvikle de scripts, der anvendes i denne forskning.

Materials

16-Channel ZIF-Clip Headstage Tucker Davis Technologies ZC16 The headstage and headstage holder may need to be changed, depending on the electrode used. https://www.tdt.com/zif-clip-digital-headstages.html
1-meter cable, ALL spring wrapped Thomas Scientific 1213F04 Any non treated petri dish will suffice. https://www.thomassci.com/Laboratory-Supplies/Cell-Culture-Dishes/_/Non-Treated-Petri-Dishes?q=petri%20dish%20cell%20culture
32-Channel ZIF-Clip Headstage Holder Tucker Davis Technologies Z-ROD32 The headstage and headstage holder may need to be changed, depending on the electrode used. https://www.tdt.com/zif-clip-digital-headstages.html
Acetone, Thinner/Extender/Cleaner, 30ml Ted Pella 16023 https://www.tedpella.com/SEMmisc_html/SEMpaint.htm#anchor16062
Baby-Mixter Hemostat Fine Science Tools 13013-14 Any curved hemostat will suffice. https://www.finescience.com/en-US/Products/Forceps-Hemostats/Hemostats/Baby-Mixter-Hemostat
Carbon Conductive Tape, Double Coated Ted Pella 16084-7 The protocol suggested three options for mounting the functional electrode to the aluminum stub (copper or carbon conductive tape or a low profile clip. We utilized the carbon conductive tape in our study. https://www.tedpella.com/semmisc_html/semadhes.htm
Corning Costar Not Treated Multiple Well Plates – 6 well Sigma Aldrich CLS3736-100EA Any non-treated 6 well plate will suffice. https://www.sigmaaldrich.com/catalog/substance/
Dumont #5 Fine Forceps Fine Science Tools 11251-30 Either this fine forceps or the vacuum pump will suffice. https://www.finescience.com/en-US/Products/Forceps-Hemostats/Dumont-Forceps/Dumont-5-Forceps/11251-30
Ethanol, 190 proof (95%), USP, Decon Labs Fisher Scientific 22-032-600 Any 95% ethanol will suffice. https://www.fishersci.com/shop/products/ethanol-190-proof-95-usp-decon-labs-10/22032600
Falcon Cell Strainer Fisher Scientific 08-771-1 https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087711
FEI, Tescan, Zeiss (also for Philips, Leo, Cambridge, Leica, CamScan), aluminum, grooved edge, Ø32mm Ted Pella 16148 Depending on the SEM machine used, you may need a different size stub. https://www.tedpella.com/SEM_html/SEMpinmount.htm#_16180
Fisherbrand Aluminum Foil, Standard-gauge roll Fisher Scientific 01-213-101 Any aluminum foil will suffice. https://www.fishersci.com/shop/products/fisherbrand-aluminum-foil-7/p-306250
Fisherbrand Low- and Tall-Form PTFE Evaporating Dishes Fisher Scientific 02-617-149 Any Teflon plate will suffice, this is used to dry the probes after washing on a surface they will not stick onto. https://www.fishersci.com/shop/products/fisherbrand-low-tall-form-ptfe-evaporating-dishes-12/p-88552
Michigan-style silicon functional electrode NeuroNexus A1x16-3mm-100-177 http://neuronexus.com/electrode-array/a1x16-3mm-100-177/
Model 1772 Universal holder KOPF Model 1772 Other stereotaxic frames and accessories will suffice. http://kopfinstruments.com/product/model-1772-universal-holder/
Model 900-U Small Animal Stereotaxic Instrument KOPF Model 900-U Other stereotaxic frames and accessories will suffice. http://kopfinstruments.com/product/model-900-small-animal-stereotaxic-instrument1/
Model 960 Electrode Manipulator with AP Slide Assembly KOPF Model 960 Other stereotaxic frames and accessories will suffice. http://kopfinstruments.com/product/model-1772-universal-holder/
Parafilm M 10cm x 76.2m (4" x 250') Ted Pella 807-5 https://www.tedpella.com/grids_html/807-2.htm
PELCO Vacuum Pick-Up System, 220V Ted Pella 520-1-220 Either this vacuum pump or the fine forceps will suffice. http://www.tedpella.com/grids_html/Vacuum-Pick-Up-Systems.htm#anchor-520
PELCO Conductive Silver Paint Ted Pella 16062 https://www.tedpella.com/SEMmisc_html/SEMpaint.htm#anchor16062
SEM FIB FEI Helios 650 Nanolab Thermo Fisher Scientific Helios G2 650 This is the specific focused ion beam and scanning electron microscope used in the protocol. The Nanobuilder software is what it comes with. If a different FIB instrument is used, it may not be completely compatible with the protocol, specifically the steps requiring the Nanobuilder software. https://www.fei.com/products/dualbeam/helios-nanolab/
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Focused Ion Beam LithographyFIBNano-architecturesMicroelectrodesBiocompatibilitySiliconMetalsPolymersNon-planar SurfacesPost-processingScanning Electron MicroscopeSEMSilver PaintStage CoordinatesNitrile GlovesNavigation CameraMicroscope User Interface
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Mahajan, S., Sharkins, J. A., Hunter, A. H., Avishai, A., Ereifej, E. S. Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes. J. Vis. Exp. (155), e60004, doi:10.3791/60004 (2020).
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