NeuralapplicationsofMEMS

1 Planar Microelectrode Arrays 1. PlanarMicroelectrodeArrays

invitro recordingfromneuronsortissue
membranestructureandpropertiesofexcitablecells
ionchannels
inchannelstructure
restingpotentialofmembrane g p
membraneelectricalmodel
patchclamp
i l l i conventionalglasspipette
microfabricatedplanarpatchclamp
2. Surfacemodification molecularpatterning p g
controlattachmentofneuronsonelectrodes
3. Implantablemicroelectrodesandmicroneedles
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invivo recordingfromneuraltissue
1
NeuralapplicationsofMEMS(1)
Learning Objectives LearningObjectives
Attheendofthissectionthestudentshouldbeableto:
describeionchannelstructureandfunction,andwhyion
channelsaretargetsfordrugresearch
describetheelectricalmodelofthemembrane
calculate the resting potential given the concentrations of the calculatetherestingpotentialgiventheconcentrationsofthe
relevantions
describetheconventionalpatchclamptechnique
l d h d l f h h h h listreasonsdrivingthedevelopmentofhigherthroughput
patchclamptechniques
describeplanarpatchclamparrays p p p y
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Furtherreading
JOHNSTON, D. & WU, S. M.S. (1994) Foundations of Cellular JOHNSTON,D.&WU,S.M. S.(1994)FoundationsofCellular
Neurophysiology,MITPress.
KANDEL E R SCHWARTZ J H & JESSELL T M (2000) KANDEL,E.R.,SCHWARTZ,J.H.&JESSELL,T.M.(2000)
PrinciplesofNeuralScience,McGrawHillMedical.
PURVES,D.,AUGUSTINE,G.J.,FITZPATRICK,D.,HALL,W.C.,
LAMANTIA,A.S.,MCNAMARA,J.O.&WILLIAMS,S.M.(Eds.)
(2004)Neuroscience,Sinauer Associates. ( ) ,
http://www.ncbi.nlm.nih.gov/books/NBK26910/
IonChannelsandtheElectricalPropertiesofMembranes
Biomedical Microsystems Meng Chapter 9: MEMS Implants
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BiomedicalMicrosystems,Meng.Chapter9:MEMSImplants
andBioelectricInterfaces
3
Excitablecells
Cellsthatcangenerateelectricalpotentials
and currents andcurrents
Thesepotentialsandcurrentscanbeobservedin
the cell interior volume across the cell thecellinteriorvolume,acrossthecell
membrane,andinthesurroundingconducting
volume.
nervecells(neurons)
musclefibers
sensoryreceptor(transducer)cells
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Neurons
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http://www.ncbi.nlm.nih.gov/books/NBK26910/figure/A2040/
Sensoryreceptorcells
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Molecular Biology of the Cell, Alberts et al.
6
Bioelectricalsystems
ManMade Electrical Systems Biological Electrical Systems Man MadeElectricalSystems
chargecarriers:electrons
withinaconductor
BiologicalElectricalSystems
chargecarriers:ionswithin
anelectrolyte
currentflowwithin
(insulated) conductors
currentflowinsideand
outside of (partially (insulated)conductors outsideof(partially
insulated)cellmembranes
insulator cellmembrane
electrolyte
Na
+
,K
+
, Cl

,Ca
2+
e

conductor
Na
+
,K
+
, Cl

,Ca
2+
electrolyte
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Typicalionconcentrations
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Foundations of Cellular Neurophysiology, J ohnston and Wu
8
Transportproteins
classified as carriers channels or transporters classifiedascarriers,channels ortransporters
passive transportthrough
l t h i l di t electrochemicalgradient
chemicalgradient
electrical gradient (membrane potential) electricalgradient(membranepotential)
active transport(=needsenergy)
ex: Na
+
/K
+
pump uses ATP
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ex:Na /K pumpusesATP
9
Ionchannels
integralmembraneprotein(canbeassembledbyseveralsubunitsorbyasingle
b i ) subunit)
spantheentiremembrane
whenopen,allowfreediffusionofions
Gatingofthechannelcanbeactivatedbyvoltageand/orspecificligandor g y g / p g
membranestretch.
Voltagedependentgatedchannelsareattheoriginofthespecificityofthe
nervouscellsandallowgenerationandpropagationofthenerveimpulse.
Ligandgatedchannelsplayafundamentalroleinsignal(chemical)transmission g g p y g ( )
andamplificationbetweencells.
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figure from Neuroscience, Purves et al
10
Ionchannels
allowionstopassthrough
themembraneunderthe
influence of concentration influenceofconcentration
andelectricpotential
gradients
selecti e permeabilit selectivepermeability
onlyallowcertainionsto
passthrough
ionchannelgatesregulate
thepermeabilityofthe
channels,allowingcontrol
overtheflowofions
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Ionchannelstructure
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Functionsofionchannels
knownbiologicalfunction
regulationofmembranepotential
signaltransduction,insulinsecretion,hormonerelease,regulationof g , , , g
cellvolume,immuneresponse
ionchanneldysfunctioncanhaveastrongimpactoncellsignallingand
cancausedisorderssuchascardiacarrhythmia.
robust assay systems for characterization robustassaysystemsforcharacterization
canscreenthousandsofcompoundsagainstionchannels
selectionoftargetsthatonlyactontheintendedtarget lesslikelyto
havesideeffects
specifically modified by and accessible to small molecular specificallymodifiedbyandaccessibletosmallmolecular
weightcompounds
genesequencinghasshownthereare>300ionchannel
genes g
targetforsmallmoleculedrugs
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Tetrodotoxin,isolatedfrompufferfish,bindstightly
d ifi ll
+
h l i ll li l
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andspecificallytoNa
+
channelsinnervecells.Aslittle
as10ng isfataltohumans.
14 http://www.ncbi.nlm.nih.gov/books/NBK22509/#A1818
l ( l ) h f d l Foxglove(Digitalispurpurea)isthesourceofdigitalis,
oneofthemostwidelyuseddrugs.Digitalis increases
theforceofcontractionofheartmuscleandisused
totreatcongestiveheartfailure. g
Digitalis inhibitstheNa
+
K
+
pump,leadingtohigher
[Na
+
]insidethecell.Thediminishedgradientresults
inslowerextrusionofCa
2+
bytheexchanger.Higher
i t ll l [C
2+
] h th bilit f di
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intracellular[Ca
2+
] enhancestheabilityofcardiac
muscletocontract.
15
http://www.ncbi.nlm.nih.gov/books/NBK22464/#A1791
Ionsflowthroughopenchannels
in excitable cells, the unequal concentration of ions inexcitablecells,theunequalconcentrationofions
intheintracellularandextracellularspacesproduces
ionflowthroughanyopenchannels.
ionsaccumulateonthemembrane,producingan
electricfieldacrossandwithinthemembranethat
willexertaforceonchargedparticleswithinion
channels.
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Ionfluxacrossthemembrane
A.InacellpermeableonlytoK
+
the
restingpotentialisgeneratedbythe
ffl f K
+
d it t ti
B.Thebuildupofchargeactstoimpede
furthereffluxofK
+
,sothateventuallyan
ilib i i h d ( h i l d
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effluxofK
+
downitsconcentration
gradient.
equilibriumisreached(chemicaland
electricaldrivingforcesareequaland
opposite).
Principles of Neural Science, Kandel et al.
17
Ioniccompositionandmembranepotential
Formostcells,sodium(Na
+
)andpotassium(K
+
)arethemostimportantionsfor
theelectricalactivity.
calcium(Ca
2+
)andchloride(Cl

)aresignificantinsomecells.
ANernstequilibrium
isachievedfora
particularionwhen
the electric field force theelectricfieldforce
balancestheforceof
theconcentration
gradientforthation,
such that the net flow suchthatthenetflow
throughanion
channeliszero.
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Nernstpotential
ThemembranepotentialatwhichK
+
ionsareinequilibriumacrossthemembrane.
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Nernstpotential
TheNernstpotentialforaparticularionisreferredto
astheequilibriumpotential.
TheequilibriumpotentialforsodiumionsisE
Na
,the
eqiuilibrium potential for potassium ions is E eqiuilibriumpotentialforpotassiumionsisE
K
.
Theequilibriumpotentialisalsocalledthereversal q p
potential.
At this potential the direction of the ionic current reverses Atthispotential,thedirectionoftheioniccurrentreverses
(frominwardstooutwardsorviceversa).
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Membranecapacitance
the lipid membrane itself has a specific thelipidmembraneitselfhasaspecific
resistanceof10
9
/cm
2
(insulator)
chargecanbuilduponeachsideofthe
membraneinregionswherethereareno
h l h h l l d channelsorwherechannelsareclosed.
b it C 0 9 F/
2
membranecapacitanceC
m
~0.9F/cm
2
.
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Electricalmodelofthemembrane
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Principles of Neural Science, Kandel et al.
22
Electricalmodelofthemembrane
passiveproperties
H d ki H l d l HodgkinHuxleymodel
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Principles of Neural Science, Kandel et al.
23
Ioniccurrents
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Restingmembranepotential
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Membraneconductance/resistanceatrest
If the membrane potential is at rest then the total Ifthemembranepotentialisatrest,thenthetotal
restingmembraneconductanceG(totalresting
membraneresistanceR)canbedeterminedfromthe )
restingvaluesoftheionicconductances.
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Patchclamp
Thepatchclamptechniqueisamethodthatallowsdirectmeasurementof
currentsthroughionchannelswithsubmillisecondresolutionusingglass
micropipettessealedagainstthecellmembrane.
The technique allows control of the electrical and chemical environment of a Thetechniqueallowscontroloftheelectricalandchemicalenvironmentofa
membraneandtheapplicationofsignallingmolecules,drugs,etc.,tobothsides
ofthemembrane.
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Patchclamp
Principles of Neural Science, Kandel et al.
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Channelgating
Ligandgatedchannelsopeninresponseto
bindingoftheligandtoitsreceptor.
Voltagegatedchannelscaneitheropenorclose
inresponsetochangesinmembranepotential.
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Otherchannelsareactivatedbystretchor
pressure.
Principles of Neural Science, Kandel et al.
29
Bindingofexogenousligands
reversible antagonist
irreversible
antagonist
reversible antagonist
endogenous
agonist
Example:thechannelis
normallyopenedbythe
bindingofanendogenous
li d ligand.
Adrugortoxinmayblockthe
bindingoftheactivatorby
eitherreversibleorirreversible
reaction.
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Gramicidin
topical antibiotic (causes hemolysis at low concentrations topicalantibiotic(causeshemolysisatlowconcentrations
cannotbeadministeredinternally)
increasesthepermeabilityofthebacterialcellwallby
allowing inorganic cations to travel through unrestricted allowinginorganiccationstotravelthroughunrestricted
destroystheiongradientbetweenthecytoplasmandthe
extracellularenvironment
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Principles of Neural Science, Kandel et al.
31
Gramicidin
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currentthroughchannelchangesproportionallywiththeelectricaldrivingforce
Principles of Neural Science, Kandel et al. 32
Recordingcurrentflowfromsingleionchannels
Ametalelectrodeincontactwiththe
electrolyteinthemicropipetteconnectsit
toanelectricalcircuitthatmeasuresthe
currentthatflowsthroughchannelsinthe
membrane under the pipette tip
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Principles of Neural Science, Kandel et al.
membraneunderthepipettetip.
33
Patchclamp
Simultaneous quadruple patch clamp recording from
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Simultaneousquadruplepatchclamprecordingfrom
synapticallyconnectedlayer5pyramidalneuronsina
corticalbrainslice.
34
Chipbasedplanarpatchclamp
Objective: to replace traditional patch electrodes Objective:toreplacetraditionalpatchelectrodes
withaplanararrayofrecordinginterfaces
miniaturizedonthesurfaceofeitherasilicon, ,
polymerorglasssubstrate.
traditionalpatchclamp:highqualitydata,butlow
throughputandhighuserskill.
i th h t i lt di increasethroughput simultaneousrecordings
d d i decreasecostperdatapoint
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Siliconnitridemembrane
positioningvesiclesonan p g
aperture(diameter~0.67
m)
thicknessofsiliconnitride
membraneh~100nm
surfaceofnitridemembrane
modifiedtoattractthe
negativelychargedvesicles g y g
measuredionchannel
currents currents
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SCHMIDT, C., MAYER, M. & VOGEL, H. (2000) A Chip-Based Biosensor for the
Functional Analysis of Single Ion Channels. Angewandte Chemie, 39, 3137-3140.
36
PDMSbasedplanarpatchclamp
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LI, X., KLEMIC, K. G., REED, M. A. & SIGWORTH, F. J . (2006) Microfluidic System for
Planar Patch Clamp Electrode Arrays. Nano Letters, 6, 815-819.
37
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LI, X., KLEMIC, K. G., REED, M. A. & SIGWORTH, F. J . (2006) Microfluidic System for
Planar Patch Clamp Electrode Arrays. Nano Letters, 6, 815-819.
38
Glassbasedplanarpatchclamp
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FERTIG, N., BLICK, R. H. & BEHRENDS, J . C. (2002) Whole Cell Patch Clamp
Recording Performed on a Planar Glass Chip. Biophysical Journal, 82, 3056-3062.
Nanion Technologies GmbH
39
Siliconbased
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