CA2352877A1 - Interface patch clamping - Google Patents

Abstract

The invention provides a novel development of the conventional patch clamp technique for measurement of whole cell electrical activity. The invention provides for one or more cell or cells to be suspended in a liquid medium at a liquid/air interface (by virtue of the effect of surface tension at the interface) whereby the cell or cells are accessible at the interface to a microstructure electrode (such as a pipette tip) to which a cell can attach to form an electrical seal, for the purpose of whole cell voltage clamp recording. According to the invention the electrode can be caused to form a high resistance electrical seal with a cell suspended in the liquid at the liquid/air interface without the need to press the cell against a solid support surface. The invention also provides apparatus for carrying out the interface patch clamp technique and control logic for operating a computer t o carry out the interface patch clamp technique.

Description

INTERFACE PATCH CLAMPING
Introduction The present invention provides a novel development of the conventional patch clamp technique. This novel technique s is referred to as the interface patch clamp method.
Voltage gated ior. charnels are potential targets for a considerable rar_ge of novel treatment~~ in a variety of disease states. The developmer_t of the patch clamp tec:~.nique has provided a powerful method for the study of io ion channel function and pharmacology in whole cells.
However, while the patch clamp technique provides a definitive method for the investigation and screenir_g of drugs with potential activity on voltage gated ion channels, the technique is currently highly dependent on i5 the skill of the operator and tends to be very slow for drug screening. The present invention provides a method for increasing the rate at which compounds may be screened for ion channel blocking/agonist activity using the patch clamp technique. The method can retain the essential 2o features of the conventional patch clamp recording system whi':~e facilitating automation of the rnajor time-consuming components of the tecalictue .
Background: Conventional Patch Clamp The success of the patch clamp techr_ique is derived from 2s the ability to form "tight" (i.e. high resistance: taiga Ohm} electrica'~ seals between an area of the cell membrane (the Patch) and the tip of a pipette. The patch clamp pipette is usually made frcm glass. 'The formation of the G-seal is dependent or_ t::e prcfile of the top of the 3o pipette, ar_d is enhaT:ced by the application of suction tc SUBSTTTLJTE SHEET (RiJLE 26) WO 00/347~6 PCT/G B99/040 i 3 the interior ~._ t .e pett~. T':e ra~:~.re ~~ts ~ -:t?
LOrmat;Cn CS ..__. G-Scal= .re we! ~ aStaDllSi-eC and ~.: c DrCC°SS 1S llS:.'.a~lV .T,C.~iltCre~ ~~~ rr~Cd~l~J ~V C~Slc.'_J C
the Current pu'ise, =eco~ae : _r_ resv'oTse ~" s _ a ma 1 '~. vci race - St°p ap lled t:~r~,llC:'lCllt Sea, 1 ni~C_ LCrma~_ P - _crmat cn. '= r ~-. cn o= a G-seal, the area of membra=:e under the pipette may be di srupted tc Obtai:: vr'_~_ole ce_ 1 ;;cltage cla;np recording mote.
The seTue_nce of events leading to successful G-seal ~o format'~or_ and wnol a cel l recordinc mode usinc pre-for~:,ed patch pipettes ~.. as =cl lows 1. Se=eCtlCn C~ a S;lltaDie Ce1!.
The L:atC~_~_ D'pett°_ 1S ~ l _ _ pCS'~"iOneapprO~Clmat°_V jC
miCrO_'1S abCVe t:'?e Cel1 .
3. The p;pette is ,~ower2d ~~ntil t.~.e cell surfa a s c i defCrm°__~, ~:~ r!-~C p;Dett'S t_D.
4. Negative pressure is applied to the interior of the Pi~eti_e L'_:a'_~ ,.. G-S°al ~S rOr;Tte'.1'' betwe°n the pipette t'_D a?':~., t_:° C°.~_y me!T'u7ra:_e.
2o S. Whola cel'~ recording mode is esta':.~,iished by the appl i caticn cf f~~rt:=er newt=-re pressure whic ~isrupts t he ce~~'_ mem:Drane in the area under the pipetto t Steps twe a nd t____~ are sic:w and rep _re considerable 2s ma =::a'~ de~ct°_ _ , and a = y ~ '~ev~e~_ cf operator ski 1 1 .
V~.Slla1=Sat_C'~ C= t_=° ~°! ! S a~,d tf'_e patC_'1 pi.pet to re~,l'._r2S
r l-o a , ~ : _.'., LSe C_ a i_____ m.ld_~~'.' c r- ~. ~ ~,..-_ _ _ "._~r0_,~.~p= and, ='1 Ord..__ LC
pCS~t_._C__ ti"1° p_p "_ ~ _ _';~~ _ ~,la__ , thr°° a:C~S
SUBSTiTTTi'E SHEET (RULE 25) micromanipu 1 atcr wit:: sub-micron: reso 1 ut;_er_ in each a:t,_s is recruired.
Summary or the I.ventior In its broadest terms the invention provides for one or s more cell or cells tc be suspended in a liquid medium at a liquid/air interface (by virtue ef the effect cf sun=ace tension. at the interface) whereby the cell or cel~_s are accessible at t'_'_~.e interface to a microstructure electrode (such as a p;pette tip) to which a cell can attach to form io an electrical seal, for the purpose of whole cell voltage clamp recording. ~.~cording to the inwent~On tine electrode can be caused to form a high resistance electrical seal with a cell suspended in the liquid at the iiquid/air interface without the need to press the cell against a is solid support surface.
Any body of liquid or column of liquic., which gives rise to a situation in which a cell cr cells are located in the liquid at a liquid/air interface, can be used in the invention. For examples cells may be suspended in a 2o column of liquid held by surface tension in a capillany tube. Alterrati~rely cells may be suspended in a droplet of liquid, which droplet may itself be suspended from or supported by a support.
It will readily be appreciate that the ir_terface patch 2s clamp technicue can be operated in "single cell mode", or could be multiplexed to operate or. a matrix of cells with multiple electrodes.
Accordir_g to one aspect c~ t:~,e ir:ventior_, interface patcri rag can utilise a patch pipette of conventional type.
o Cells are supported on a lic-.:~.d/air irate-face at ore end SUBSTITUTE SHEET (RULE 26) a CaD1'.~~ar' __'~~E' ~e r, _ , y mace O. i ~ as= ~ p01'Jet :~ 1 ere C_ Ot:"-er .. ._....~._~ :;?c.~er;.._ , . T':~.e a:C~ "~?' S ~, t=:e C.atCh i~;~ett~
iS ~ : 1 i=:e ',v;t__ the =___.. ~._ the t',=be sG t:at tile pi pette _ '' 'y'e "~ ~ ~at'~u .~... t_1e ' ~~~ C3__ ma___~;; __.. ODe_~__~ g C~ t .e t;lbe Gl:lere S t.~:~' Ce.lS a~e S~.;C~C~ t'~ ._,~ ti':e a~~"; 1 '_~u;C ~='_terfdCe . i'__e CaDl.lar_J tube Gr the DatCh DiDette Ca i1 b2 mCllTlted Oi:tO a SlPgl a d 'r.s mc_Tl'~.~,l.',~ ' Gr -, _at . v; ;_J O:-a T'.':al_1~=.ul atCr 1S re~_Ll~rred and t_-~is ma_~ be used tG .'~.c~,-e either th=
patch pipette or ti:e Cap'_i~ 3r :' r'.~Ce . T/~!i?c~ a _e! ' reCCr'"l~g mCCe 1S
est3b11Si':ed as LCI~~GWS:
5. A layer CL Ce_ls '~3 e..t3~~l;S~':e~ at t~"'_e '_PterfaCe between r~e °_~traCel~~ ~h "' ---- _ _.'S .GlOC~ Ca_ sOllit'.:.OP_ (the liGTllid i_~_ ta.~_1Ch tl:e Cel 1S are SL:SDe=lde'al a?'ld a1~ ~.''V_ dippl.= J the cap il ar'~ _ ;:,'C
'~:be ~ _ a sLaper_s,~Gr of 1~ Cel 1 s . '_T're de:'?si tV C~ Cell ':.
s ..h a s us~ea'i Gn Tus t be suf'ic_e~t tc ~ wide ~ sufric~er-- .. __'~mber o~ cells r to fc_-n a ~;a~rer or ce=is at the interface.
_leCt=lCal C.~._.~_taCt :v~th t .e eYtraCel~~ll~ ar ,,lt=Cri 1 SCl! S
established via a nCP-polaricable electrcde (e ~ an 2o Ag Ag~.1 Wire) ar_d the tube is mcunted t i...i t0 a ilXeG C_am~ Gr S1~~~le aa_S may=DlllatO_".
8. A patch pipette is provided which ca.~_ be filled with electro'l,.te solut; -' _ _c:_.
'~'he patch ~' tt,~
..,ipe _ _~ moLlilteC C~~.TCe?1tr_Ca~ 1'J With t_'1e zs capill ar-y tu': a eit_:er -~ is a sing 1 a a~cis manv_puiatcr Or ~ l::ed C , t :_ '-amp ( __ ,.__~ c~p'_11 ary t~.:be is to be moved) . _.e pi~ette ri,.~l i _g so--',~e~ 'is ccrr_ected via t':=a ~on_oeia___~~_~ e-_ectrcde a ..e _ tc th aasta a co:_-re=:t_c:.a~- pat..__ ~ =-;;--_.- _ ~. _ ____~_ . The ~'~~ett_ ~o , : __ _ .
:holder al_cws sLCt«._: t~. be app'_-ed to t__e pvpetta inter~"_.
SUBSTTTUTE SHEET (RULE 26) 10. Cell attached patch mode of reccrding is established by bringing the pipette tip in contact wit:: the interface by moving the pipette ar~d the capiliar_r tube respectively together along the single mounting s axis (e.g. either by moving the pipette towards the t~,ibe and interface or vice versa). On entry into the interface the mo~fement of the pipette and capillary tube together is stopped and the pipette current is offset to zero on the patch clamp amplifier. The ,_o resistance cf the pipette increases when the pipette contacts or_e of the cells at the ai r/1 iii d interface. Sucticn is then applied to the interior of the pipette and the pipette and capillary tube are moved closer together until the pipette tip is i5 located inside the capillary tube.
Initial seal formation between the pipette tip and the cell may also be assisted by the application of gentle suction during entry of the pipette into the interface.
zo A G-seal is formed between the patch pipette tip and the cell membrane by the application of further suction to the interior of the pipette and mor_itoring the pipette resistance.
11. Following the formation of cell attached patch mode, z5 the suction is released, pipette current is offset to zero and a holding voltage appl=:ed to the pipette (e. g. -60mV).
I%. a whole cell racordir_g is obtained by the applicatior_ of furtrer sv.~c~,ion to the pipette interior until the ;o whole cell recc~di~.g mode is es~ab,~,ished in convent;~opal manner.
SUBSTTI'U'TE SHEET (RULE 26) WO 00/3.1776 PCTiG B99/04073 ACCOrdlng LC triS L::JenL_~__ _~ .~~ - 'ferrSd th- tl~,~.
~t CaD~i!a~V L::"JC SI-'_OL~~'~ .~_ iLIGUTlt°C L__ aW .ipr=QI:.r.
Or~'=ltatiCT1 ( i . a . esse-tia~.'_v~ -:ert~..~ii,,~; w'_t'_-,_ the a_y!~~.c~:;vd interface at t_~_e dO~,~rr:ward end of t=,e tube.
s This has the advantag: t.~:at susper_ced cells -~n'~,~ tend to ~~sedi~le=-t" :'atL;~d'~~:' t0 t::e CC;Ji.~.Ja~.'~. ei!d CL '"~F' '~,P a-,d t__,. t'.:_.
L7e COIL eCt°d t~'lere _ r __ 3 aye . _~~.' layer w-~_ ~~e'e_abl'i be several cells deep and loesel;r packer. '"hus according t0 the i'~,~J°,.lt~ ~-,n t_:a .~,L'~°t.~? t-~ m,aV ~bc mOtIcC ~l ~.
_ L: N ~lJ a r ~.. _. r 0 relatlTe t0 t~° a-"'~ZiG'L'.~.1.~ Ln tC~'riaCe a~ t~e s.:iDe T~ _ e__d (either by me~~ir_g t'.~_e ~ioette or t:_e tube alcng the singe axis? so as to come irte contact with a cel~ in the gayer at the '_nterfaCe. '_'!''_e ra',_at'~sle CenSit:v' Or COnCentratl0;'_ Of Cells at t__~ in Le«aCe COm;,ared t0 th a denSi tV 1the 's bulk of the lya'_d ~_~ .he tube a~aures a '~;gh probability that a Cel l Can be COI IeCt°~ Cn the t1p wit_~,OUt t~'1e nC2d~
LOr ViSUaI lSCt'~O:". O_ t~_e C~eratlOW and With ,~ -,cep _ ~'.a tr_' ~_ fOr mu l tid=reCt'~Ona~ f~la_T':lCulatlC_~. CL the t'_,,.'~% Cell pOSltlOnal ~elat'~C_~_S_'1'~.D. ~tir~r i' _ is__~giy it has been found i 2c that G-sea_ fcrma'~_,:n between the cell ar_d the pipette can occur without pressing t.~_e ce,l aJa'_nst a se'_id substrate.
where the arrangement is -lr.tended to operate ~,vitn the plpettC In a:WDriQht CrieWtatiOn (l. e. esse_ntlally vertica_?y; w'_r'.~_ t.~_e t=p uppermost a nd pointing upwardly, 25 the pipette S~'!0~;~d be C.~.nSt?"'~:Cte'~ c0 dS tC 'TJ~eVe=1t th°
flll~ng el°CtrOlyte SOlut=On LlOWlng Out dnd L~elll~7 IvSt.
This may be ac.~_ie~red for exa:rol a by use o= a custom-made mounting assembly a_. jer ~,_i shapir_J t he pipette body to prevent less c~ Lill_~G sclw~=cr, (e_g by bending the 3o pipette s::at i nto a ~~~- c~ ~ s:tane SUBSTITC1TE SHEET (RULE 26) The invention also provides methods and apparatus employing control logic to allow automation of a patch cla«,p system employing the Interface Patch C'~amp tecr~ icue described herein. The logic described w=11 control one or more electromechanical micrcmanipulatcrs/translators holding one or more patch clamp pipettes and/or capillary tubes in order to patch clamp cells azd apply drugs/cempounds in order to screen for activity en membrane ion channels. A major advantage of the logic io described is that automation is achieved in this system bar the use or feedbac'.~c from sigr_als from the patch clamp amplifier and no image recognition software is re~:ired.
The invention is illustrated by wav of example in the accompanying figures in which:
is Figure la shows a capillary tube containing a susper_sion of cells; and Figure lb shows the cells having formed a layer at the air/licruid interface at one end ef th.e capillary tube;
Figure 2 spews a general arrangement of the interface 2o patch clamp recording equipment with moveable capillary tube;
Figure 2a shows an apparatus for Interface Patch Clamping with drug/compound application;
Figure 3 shows the cell attached to the patch pipette 2s ready for recording mode.
Figure 4 shows drugjcomDOUnd addition during interface patch clamp recording: start position;
Figure 5 shows drug/compound addition during interface patch clamp recording: extracellu~lar solution added to 3o dish and d~~sh moved down;
Figure 6 shows drugjccmnound addition during interface patch clamp recording: solution in dish brought into cor_tact wit h interlace recic.~.;
SUBSTTTUTE SHEET (RITLE 26) PCT;'G B99/04073 _ gore % .~-.I1CWS C~'.:C,'.~..~.:1: CU_~_C aCG~L.C__ C'.::rlT''_g ~_.tC=LaCe oatc__ cl amp reccrdi~ c=-' ~r-.
_ _ _---_,~ r_ised a;~ove sur=ace e=
soiucicn in ~_~_ , t'~Ciire C ~.J a __v... C=aJ ....,. C. u.u_"_t=C; ~~OC~ ~ ..",'.OCC"..'Ji.lG a .. iur '~~er avLue.~.s. v_. t.:e __. J ~?" ;. rlT, .
__l.~W _, Figure 9 is a _,_cw diagram o' an e:ca.~.;n~~e o~ the G-seal formation steps c~ t gore g;
Figures i0 to _~ are ~lowc~arts ct a t__=rd e;nbod;me~_t oy tr:e ccntre-~ i cg,~c used i _~_ the i~.-Je:~!ticn , -NCerei:.
1~ F' Qure _.~ ~S a ~_C'N C'iagram C_ Control _cg=C aT,~,YCCVInG a ~urther aspect c~ t he i nVentc:., F ~ pure ~ Oa is a fl ow a -- routine d'._aCra,T: xrJa:'idlPJ tr?e reset oz figure = , F i~'~r... i0v7 i S a fl OW diagraT, a ~ua=:C.'~nC1 Lne Sage Cjult __ rCUt;ne C
figure ;._ .S a Ll .~aC
C'~L' C ram eYpanCinQ t :e 1=;te?'LaCe Or Ce~~1 lCCatOr ~Cllt~:!e C~ L~Jur°
Figure ~'3 ij ;..~CW C~agra:';t eY~'and~!'1Q toe interLaCe r?t rCUt'_ne Ct '~Q;=re ~ ~ , 2~ Figure ~ ~ =S a t_CW CiaCr.... . =:C ~' . : a:lCl'~ t:=a ~ ~ Ca Sea= test r routine O'~ iiC,'L:r°_ ~ C;
Figu=a 13 _.. a Llow diagram e:~pandig tde whole cell detection rcut_ne c~ _iau=> ; , Figure _ ~ _ ,~ =-'_ s a slew diagram e:cpanding the cuaiificati or:
2s routine Of = c"r' 1~;
Figure =5 is a ticw diagram e:c~arding t__~. qt;ality monitor rout,_ne of _ J re __w;
Figure 15 is a L,_cw diagram e:cpand,;_~_g the e::periment routine o~ _igure 10;
3o F figure 16a is a 'low diacram e:~nandi ng t:e run trace rGUt'~:le CL =
F ~ gore __~ __ _ __o~N C_acra:v ~ -~~:_ g ho ' i .._ .~.a__._~ i t S a~1 1 Se rCut_ne C= L=gore ~" ~, _ eS _ . ~., _ ~ =C'N reCCrC=:=CS cL C'_r=2n t ( l i a_~_d VCltaCe ( CC=_ ___ .. __ ,. ...__ cutClilated D3tCY: ClamT~ S;'~tem SUBSTITUTE SHEET (RULE 26) _ g -(AutoPatch; under software control usir_a the Interface Patch Clamp technicue. (~cecording from an MKl cell);
_,-gore 2. shows the e~fect of t:~;e potassium channel bl ocking drug tetraethyl ammonium (TR'=..) er_ t'ne aotassium s current recorded from MKi cell in whole cell recording mode obtained using the Interface Patch Clamp technique;
Figure 22 shows cells suspended in a droplet of liquid covering a hole througr. a support surface and providing an air/liquid interface according tc the invention;
io Figure 23 shows a further embodiment of droplets containing cell s~,~snensier_s forming air/liquid interfaces according to the invention;
Figure 24 shows a multiplexed ~rterface pate:; clamping array;
~s Figure 25 shows an alternative form of eectrode microstructure according tc the inver_ticn; and Figures 2o'a ar_d b show a multiwell arrangement for drug application to patch-clamped cells.
Referring to Figure la; a capillary tube (1) of 2o appropriate size can pickup and hold a liquid sample (2) containing cells (3) in suspension. The sample can be picked up simply by dipping the tube end into a suitable bulk lic,~:id reservoir . The li ouid i_A the tube forms an air/liquid interface (4) at the tube end (5). The cells 2s are initially distributed throughout the liquid relatively evenly.
Referring to Figure 1b; with the tube in an upright essentially vertical orientation, the cells tend to sediment and to pack loosely together at the lower end cf 3o the tube by the tube end to form a l~;var (6) several cells deep. It will be appreciated by tho~~e skilled in the art that the density and depth of t::e cel.l laver can be deter-v.ined by such factors as the ce~.i conc~ntratien = n SiJBSTTTUTE SHEET (RULE 26) t:'le Cr"~ginal S'~:5~erci Or. tye SeClT" =CatlCn t~.::e, t=:e rel atirre densi-_r c_ ~:~,e c=1 , ' tva , etc . It ~~=1 c_r_~ __ _ i ~l:=. d 1 s:lSO Le aL'vrec a t~~:aC ~~.ea j.ate . .S CCi.:_.~.. De Cev''-SeQ t..
enCOuraCe Gr aSS~S= =-1' c~ __ tC m'_graCC ~rOm tile ~~~_~:iC
s towards the a,;rjl' =c'~-c __ace rac:~er t'',an or as well as re l ying on gravi tational sedi~':entation alone . '_''he F
also shows the top of a patch picette -~ pointir:g upwardly towards th a irater=ace.
Referring to Figure c; an arrangemea is 5hecvn i whic:_ a lc single ails mar_ipulator ~_ use~
<i.ove a caoz~; 1 arv tube _ field irl a clamp (%; re'~ati-re to a =___ed p=tc'o pipette (8' - _ helG' 1n a Clamp (~) . I t W~ ~l be ~Cpare~t t0 thCSe Skl1 led the ar t that .n~ 5 ~~..1 ~ ~'a',r'r-c be _ . ~ed so that the tzmette is moved an d to tube is t ~he fixed. _ _iaure shows the tube ~s clamped ir_ a linear bearing sl_...=rag blcc:~ (lei attached to a motorised sv~ngle axis ma~:inulator (1=). The mar_in _w u~~~for should be ccr_troll ed prefera': lv .~.v~ computer ir_ order to allow the moticr_ of t::e :~anin,~';atcr to be varied by feedbac;~ frem tr_e pate:: clamp ampli=-~er . The pate?:
2e pipette is provided with a ccr_nec~ion (12) to a CCP_VentlOP_al headStage. Tile 5..'Ste~T, iS a~'~.SO 'Jr0'rldeCl With a source of va~riabie suction under t~:~:e control of the patch c'~amn amp''ifier/ccmouter.
In Figure 2a an arrangemer:t ~~s shown in which addit,_or:a1 zs a 1 ectrcmec~ a. -.cal ~~:ic=oma nipulatcrs have beer added. The mlCrOma_llaul atCr lafJe l led (? ~ . __ ~Oi mCVinC t.ne glaSS
capillary under a'.,:tomated cr r.'.a n'~a- ccr_trol . a second micromanipuiator ( 1 -'_'_ ) moves the ,:_~.. for dr ug accl icatior:
up arid dOw:'1 the g~~a~J caC=1 ~'~Gry. ~_ _____~. mlcr~~.~ma_~.~Dlliaw:_ ze (~J) moves a mcdv;=red ci~ette ~~c to provide elec-r~,_,.__ _ h~_ ~_ cCntaCt W~t h t~:e plDette a=_~ a mecn~= aDDlV'~r C SuC_ O~ _ _ _ ~ '-C==
tc the ir_ter _c_ ,._ t_.e -,; oette . ...."tional bases '' ,.
a . C_ a ~ ~ OW t=:e ' ~e~ ~... '-i ,-''a"
_ _ ~_o ~u _ to be moved ~__ ar_d cu : cL the SUBSTTTUTE SHEET (RULE 26) recording area and rotation of the pipette through 180 degrees for fil'~ina with pipette solut~ior_.
The Figure also shows additional features, namely; a pipette holder (18); a patch clamp headstage (19); and a s dish holder (20).
A version of the apparatus is envisaged ir_ which patch pipettes will be loaded and filled automatically under software cor_trol. It is envisaged also that the loading of capillar;r class into th= apparatus and the filling with io cell suspension will also be automated.
Referring to Figure 3; a G-sealed cel~ 3 is shown held on the tip of the patch pipette 8 and positioned within the entrapped lia~sid volume in the tube.
Cell attached patch ar_d whole cell (voltage clamp) is recording mar then be tamed out .
The invention described herein has a number of significant features:
~ Visualisaticn of the pipette and the cell is not ao recrai red.
~ Novel recording configuration that would not be considered as obvious.
~ Surprisir_giy G-seal formation occurs without pressing the cell against a hard substrate.
zs ~ Cells =orm a layer at the solution-air interface.
~ G-seal formaticn may be achieved using electronic feedbac:~ a';or_e.
~ There is r_o re~~iremera for optical recogn i t ic~_/ f eedback .
30 ~ The system ca=~ be automated .
SUBSTITUTE SHEET (RULE 26) WO 00/347~6 PC?/GB99/040 r3 v~.~ltic_~ recoro__na ca~
_ _ __.._~.es a;:d pipettes --; be empl0 ~ e,.. ___ Cr :e_ ~~ ci '! OW reCC_.~.1:1~ t.., ~_ TaQe Slmul to neOllS1=J =_..,.. ...~.~_V Ce=_~ .
XemplarV met:'1CCS C= perc',~_Cn O~ _:":e apCaratllS O_ th2 emh'OQlment llnCer SCLtSJare COntrCl tC aChieVe VaripllS Oi t:"leSe aQVan tageS are C2SCr i beCl bel CW .
In order to use the ___~.rentiCn for screeni~ c ~ I
-.~ c mpoLnd , a . g .
fOr iOn Clla?'Lrle~_ ~J~lOC.'C1I1Q/3C0:11SC aCtlVitV) t~e COfT'.TJOU:?Q Of ~1:~._.ereS~ ~GG~~ ~C ~~ 'v.r~ ~ __~ _.~ t._s vel I .~_, ._ t t a neC t'C t ne is pate:: pipette. ~t lr~v;~._ _~~di~v d a app eciated that this C..,.._'~Q De a~___~.
nW r-.~- ~-r.'C it !~~==erer!.' 'riaVS, LCr e:ramp'1= DV aCl,:"~.r.Q
the ccmpeund tc the e::tracel lul ar liquid ~__ the ca~iliarV
tube either beLOre or a=.
'er ~-seal formation. One aCC~,tiOna_ 3QVarW aGe C_ t.~_e =n'v'e="!t~_OI? 1S that Che liQ1.11d 1~ In t:'le tur'e C..,_''_C 'Ce _ (' ~' 1 ~.-a I
_ ar_Je~ ___ c~ r_rS ~e.G~'. :Cllt~ui_~i . ng di ffere nt ..cmpounds cr dirt°_ rt o a o=
re_ c rc ntrations compounds) a_nd the si~.c'~e a.:is manipulator could ther_ be 'lSed tC Cr:VSiCar_1V ;T!O~re aI=C ~;OSitlCn a C~ 1 CI: a p'~~etCe tip 1_~_t0 a C!'!CSe_~_ ~aVer i°.C. CV lT:OVInCJ, tile C-SealeQ Ce'1i 20 On the tiD r';.:r t_:er t.':e t;~be aW V G ' way from th~ ai r/licruic _nter lace at cr_e t~~:~ ~ en4~ .
A further examc~.e of hoW the effects ef compounds maV De studied is ~-lustr~ted ir_ figures 4 to 7.
Figure 4 sh o~ns a cap,illar-_r (1) containing the cell SuSpenSlCn (2) and DatCh C,'~pette (~) 1n the reCCr'ding pOSltlCn fCr ~ni nCle Ce''_~ reCCrdlrg Lrom a Cell at th a y.-v.lt)eCCe tiC. =dd' t G
_ ___ ~ ___"_ , .'1~ Ca.~.li.ar',J tllDe haS been nserte t~_~_rCUg h ~. ;~ dish , ~) (e.~
._.._~ _ ~:.,dde ___ a 35mm plastic ~~_tur' disc c_ ,a,-) ~imi_ . The dish is made 3C r,L ca T.aCeri a~ NW t._ _ ._ ru.~_~..~,_.. _ "CertleS anC t__.._ P_Ci°_ SUBSTITLrTE SHEET (RULE 26) - ~3 -allows the dish to be raised and lowered along the ax_s of the capillary by means of a micromanipulatcr (14).
Figure 5 shows the dish after ;~ has been filled with extracellular physic':ogicai solution (23?, which may s contain the drug to be studied, or the drug may be added at a later stage. Surpr,_singly, if the fluid level in the dish is low, leakage through the hole does pct occur because the tendency to leak is counterbalanced by:
1. The surface tensicn of the water l0 2. The attraction c= the water/sclution to the glass capillary.
r'~f te?~ addlng the SOl ut 1Cn t0 t he Q~SI1, I t i S lCWercd In the direction of the arrow.
Figure 6 shows the so'~_ution in the dish in contact with ~s the end of the class capillary and the patch pipette. The dish and the capillary are now raised simultaneously (arrows R and B) in order to pcsition the pipette tip/cell within the '!aver of iicuid in the dish. If drug is present in the dish at this point ar_d. the capillary and 20 dish were moved upwards rapidly, this would constitute a rapid application system particularly useful for the study of agonist responses that desensitise.
Figure 7 shows the effect of raising the capillary so that it is not in cor_tact with the liauid in the dish. The 2s pipette tip/cell remains immersed in the external solution layer in the dish. The soluticn may be exchanged readily by perfusicn of the dish and this allows multiple drug additions and dcse respcrse cur-res to be obtained while recording from the er_e cell.
SUBSTTTITTE SHEET (RULE Z6) Contro' Locic =cr an ?'.:tomated Patch Clamp System ntrcduc =c--a tcl lowinc descriLes th =ee e~~.bodiments o~ the cc:-_tro' lOglC reCl:ired trJ dl 1CW alltCmat';Cn OL d patCn C_Gmp S_vSte!Tl s emplcying the I_~.:.er~ace -.?~ ,-' Pat, ~iwmp techni~~e descrioed herein. r_n eac.r, cases, the logic described ,.ril .. rol _ cr_t ene or more e~ectrOmec~w _ __ : a microman'~pu-atcrs; tra nslators i:: ..rder tc pa' ' c 1 amp c:~
cells ar_d a -r pply a_~.:1 ~~, pounds ___ ..=cer to screer_ _~_ lU 3Ct_Vlt~Y Oi_ ~''.e.:.=ra~.- ~ ~~_ O~'t~i: iEIS . =_ ma ;Oi ~C'Ic'tI!twGe O'_ the lcQ~c d=SCrib2d is ~ ---t _ av~tcr:ati..__ is ach ieved in t:=is System bV C .°_ llSe C. =°°_C_GaC;~ fr :i:, S~g-:dllS
LrOm t~_'?e ''~citCr Cldmp amDl~t'_e" anQ ~C ' =o"
- _. _:~dCe '~.Cg:~:-t_OP SOlt'.JGre '_S
reauired.
is MAthods :1DLLS tC tie = "'~Q_r,~..:~ ~_. .:~_ ~- :'e e:i.:'CCC~1!IlC_lt:S Gee reC'LlYEu ty0lll thS ,'JaCC : ~~d~"~ ~'~=Sr = G:'::D__ _ _ cS ~C,!~CWS:
Imon = current mcr_it~r Cutout Vhoid = hoid-~::c pctent;,~_ 20 r_~.L'L LS t0 t -'e Cr~~qrd;rl Qcrl IeC i=C:~I Ca=C:: CIaIPL~ ct:lIISi?Li'i Oilt.~Cit 51 Q~ __. :~YC r _ _r°C dS =J~_CTWS:
Inoise = base 1_ne cur____~ noire recorded from Imon Rp;~p = pipette resistance Rtct - '='oral _ ~__~ ~a nc=
2s Rs derived L
_... __ _ J _~_ a.~_i~J ~~_tage step It is en~.r~.sace~ t:.a~
_ _ _~ ~ gna~~-s a=:c e-,-aluated values 'Nli~ be 0~.'~.t,=____ _ __CT. C~C' c' C,-' __. __ y S t'_riare '.SllC_r' ~J ~'~.'l3 SUBSTITUTE SF~E~T (RULE 26) se=aware) v=a a suitale so_tware in-eryace. T'~ese S1~='lalS aiW eVal'::ateC V?._ileS aYe = _.~ W~~.r Ce=~~='!eC1 -:1 tale 1 ' St C~ 'v'aY'=a~le5 a =C pa.a:;lete=S Je~_OW .
llllJUtS ~rOm mc~P'DUZdCOI~S~tr.iiS~~'~CrS are :e~ulrcC aS
s follows, or ircm the ~oliowirg devices.
Patch module micromanipulator encoder Capiilar-~ clamp/Ioader encode: and empty signal Pipette automated clamp/loader encoder and empty si~r,al Two axis translator encoders ror cell dlDDer Drug ao_ plication micromanipulator encoder Pipette holder micromanipuiator encoder Cor:trc_' outputs trom computer are =ecuired 'or the iO~~OWlng deVlOeS.
Patch module manipulator Pipette automated holder Capilla~--v loader/clarnp_ Pipette loader/clamp SUBSTITUTE SHEET (RULE 26) WO 00/34776 PCT,'G899104073 Tv'v0 :!.\iS C:.Ii:Si~i~.~ ..~, ~..._ ........_~ ~ ~'v'J~~:~7 Po°ne c:a.~.m ~ _ Suction cevic°
Drug ~pc;icat:or: .~:~,~,iTul,~:~or D: u° ~2.'::S1O.~: Sv!v.~.~a~ V,~.i'.'? ;'v'j;°:~.
c SC=~'ad~e t=SeS S'~_a!S ,____'.~'e:. __ 'l"'a t, "'Cm ~:: DGtC.~ Cia, am~i _=i e~ ___ .._ er tc ~ .,._,.=ci a r_wm.~.~er or oe= ~,e,- - _ _ _ a_ devices whic__ ~~_ c l.:r;~i~a usi::c L:_~ ___~__=ace Pacc:~ C=amp ~~,~__._ _ ... ____ de-r,.'-c~_ cCr_crolle b~,- t~_ .. ,~eg=c ccm~.rise a ~:wmoe_ __ ~,:ic_c~:: _ ' w _ , a succucn de-ice =cr c:-~e ~ ____ _ _ _~__ ._--:d a -,~_-re s~~rsteM~ =c.
De==llS'_C_1 ~_ ... __~.~._~__ J C:'lam;,"e= . "._. 3S '; a .~.
C ~_ 5 CSSC~ '_DeC a C'v a .
l _n_',-~e~ C' :-a=:..;lete~S a~°_ Q'.~'ieiP ~ _-Set 'v'a~.:e C a.
i t S WnlC
_G be C_r':a?~:CaC D:% ~___ ODe~_.~.._ ", SL:_C _ ' '~er'- ~XNer-.., - _ __ _ ~ _...enta!
COi_C=~~C~1S .
Sumr,.a~~ of t he coatroi icc_c =~_ the aute:nated T_nterface Patch Camp - Fig s t ~;.:~cdimer.r_ _nitial seal =~=matic-I ha Se.'.:e~Ce .';_ ,i?C've:'lCDtS ~eC"=.~'r.~~. _',_ SC=TTlat~.:~~: O= a ~:-SCal 1S lln~~,le =.._ =:?C.e='aCe paCCh ' ~~~
'.a.. w g an d invcives tPe CC-:t~C .r_ ?.. IeaJs OTe c~~g1C c::''~J ~T~aI-'_~Di_1.t0= ~e.C~'.
vawc_z mca'~_' Tc;~~ , ~__ :o'cgr_ e~_t;:e_ ____ p;pe~ y ._ a C ~.1'1 C~v ~ ~ '~1..___~i ~(a~~i ..~_ i'.~lCve~ ~C ~GC_rl_:.'~''i ° ~.
'~' 1 a _ : a ~ ._ ~~ a J S a = ,f' _ 'u. 1 TJ' a 2o movemer_t ~et~.veen t em) wit = fea~,~aok ==om t~"e patch camp am:~_~r~e=. _.. a L____ em., ~;mo--_ Ou.._ ~.__u. C '~._'=a r' r._~~
_ C.,n__.._ .~Cg=C, plpet to -S __. _ ._ -~~ ~ ' _ _ _ _ _ _ .._ ___,.., ~___ CdDl~~a=V, _.r ''~~~~~_._=~te'~ __. _ J _' _,. _J_ '_.a;;lD_ d~C~., ~S -.T~CVeC ~C~,J?=dS
r._r!a 1 1.:"..._d~a~= ,.~. .. '-,o ...
_ ___ __.. _. _... __. ... __~ ~ 'I ~Y1 _ ~ L. ~ ~ 'v. ~ ~. _.
_._ '~~w:Ge ___ t_1_ _.u__~___ .....___~'._ _. _ _.,._ _s _.w.~._'~~..'e,~_.
'.'fi DG..C_~_ p~ ve.~...._ _.__~
_ _ _~ ___~ ~._ - ~~, ~__ _._',-e«...~.v. a l~.. 1,.:_W
SUBSTITUTE SHEET (RULE 26) signal is used as the trieger to stop the micromanipulator. The pipette resistance may be derived from the output of the patch clamp amplifier and initial seal formation is mcnitered by recording the change in s pipette resistar_ce. If the resistance of the pipette does not increase beyond a pre-set value, t_he control lcgic inters that no G-sea. has been formed and activates the patch module motor to move the liquid;air interface and the pipette apart u.:~.til the resistar_ce increases, which to may occur when the pipette tip is withdrawn from the liquid or when a narrow neck of liquid is drawn cat by surface ter_sion between the pipette tip and the capillary end. When a resistance increase to a pre-set value is recorded suction is applied tc the interior ci the patc:~
is pipette and the patch pipette and the liquid/air interface are moved towards each other to a pre-set point, in a further attempt to form a G-seal with a cell.
Whole cell recording mode after formaticn ef the cell attached catch clamp recording 2o mode, whole cell mode is obtained by t:he application of suction to the inter;_or of t:=a patch pipette while simultaneously monitoring the current (Imon) roc capacitative transients. In the logic: described, the formation of whole cell recoiling mode is detected by a 2s threshold crossing method but it will be apparent to those skilled in the art that other methods may also be emnloved e.g. onlir_e FFT (Fast Fourier Transfcrm), Templara ~latchina etc. The control logic checks for incorrect detection of whole cell mode before activating the 3o experimental protocol.
Cel 1 aualit->~ test SUBSTTTU'TE SHEET (RULE 26) WO 00/34??6 PCT/GB99/O40?3 - -_s -T(I1S rOUCIn.", iT.CnItcrS Lh° .~,1_".::1C'.' Oi ~
'Cl...=_ .,...1:.~ .~~' COrnDa.in~ t~C SerIeS r°COr~OC
resistance 'With a value related to oice:Le resistar:c°. a will be apprec~a a~ -~ ~' ~h _ ~ - _ _ . I~~ .rat ~. a rr:e: od l~la V ~ -frPm ° .
ce lt:l ... ennanc,. ev -_..:I:.I~ the acc ;~n~ -,e _ _ _ ~ .e se.: s resistance Co t:':e a_,:o.!tuce o,~
current evoked by a v~oitaQ° scec. In a~ ~~Li~n a _ ~e; ,. . an c,~l i,.nai toe,, may be added Lo ~:::clude the possibility Or recordirl~ w:t:~: a maintained ie~:e' i suction a~ n .
o ..p.!ed ;o t~:~ ~laett" :n Cells L haC C,",r:lCll COIIIInli0L:51.~ !rIC'°a SIr.~T ~, a.'L:CS
OSs:'.eS reSiSLa:.C°. T!:e -;,;laIL'v' OI Il:.a, cell is also monitored oy Cl:e hcidin= cur:en; 'v'',:ich si-loud not be r~.ore negative :ha.-: a pre-seI value. it will be apprec:at?d tct ;;~:s r-I°L~:od could be enhances by relating L':e ac:.eptable value 'or hold:n~w,e;:! is ;~:e a~:pi:tude ethe current in -esronse to~a voltage slec.
Dru~/co:~Irour:d acoiication 1 ne !nit:cr .~.ra'v'. C: s.rlc nn~':..,.::L~: .~ ~.. __... ":i,'_rlaW ~.'1 "
...-..~ .
. , _ ...~ .....~::1C_'~., i!;~
lnVOjVeS Lhe COnu01 OI tW0 S!"~~~ aX!S r:~iC' ~r:Ia~~Oy!-,rte '~
:c . . ~ ~,rs. l;Ie move,: enls r°~ulred utilise the pCSlt!On OI Che Ca;::': :".lOCI!i° :r:_ICrOmar:l(;?UlatOr reCOrCeC On C.''.C:~~ :n.CC' Che lnLeriaCe aS ' relerenCe OO:n;. =..ter ::".' C°!: l:aS ~~Cen 1r:1:"?erSeC InCO SOlUCIOn COnCa! nod In a perILSiOn C(:a.:OCr. Che ;:On::C' :o':C :.a::~ .. .'~~-C;!r.'' LO Cali_, Ot:' CerI;lS';On Oi ;he C::~.aIr:ber Vla the aC;l'vatl0~ Ct SOie~0:~~ ::W'W ;.C:;;:~,1 ~ i 'eS. .
~ a. V
Control Loaic in detail _ ,~iT;~ ~_.:cdi:ne-_:L) VariableSlpara~neterS
P = Pipette pressure relative Io at:rosrh°ric p~°ssure delved as 0 d = Patch module moor pos:t:cr:
d0 = Patch mod'~le rr_otcr star ~os:tior dl = Patch module motor position i:ollowinQ entry of pipette into interlace - p ' d_ - . aCC.': mCQLIe mOCOr DOSIt:Or: W'I;h Clpet;e In .'CCOr~:n~ pOSIIlOn d~ = Patch n :edule motor position 'or c::am~e; c~e:usion dapp = ~r,~~ applica;ion I~~.cduie mic:o...anin~later :,osition dapplC. = IJP.1~ apCllC.''.ClCn :=':CC'~1C T!C.'Ci'.I'ni~UiCCOr pOSiClOn CapDl = dP.la 3~G11C::C:On .T~~C:~:'lanl~L:a:Or COSICIO!1 !nCr°.i.enl 1 Pre-Set increment dapp- = drug application :~:ic:cr.-:an:reiater posit:', n ir:cr°ment ~
Pre-se: increment RS - S~i';2S ~2S~S~3::C2 .
CJ1CW' = SOW W~c~.~_~._.~._ ~.,. ....,_~_~..
CIaSt = inSL C3_.~JdC..L__~;C°_ ~..,:.~.C~.~.S~~iJi:
Inoise = base lire noise Rein = pipette resislan;.°
R;cL = Total resistr.c°
Rl Intial seal :°sislan.,e i ~=e-~s='.,y~el R~ Seal resis;a:~c° . ~'.:i:-.... e: fro'-°s.;:o;. r ~,~;~
,°Il . ~; ',~ .. .. ;.
SUBSTITUTE S~iEET (RULE 26) Vhold = Pre se: holding potential in :~_V
Imon = curent monitor outt_:ut i = pre-set holding current Ihold base line hoidina current fcr hciding potential dpip = pipette holder module tractor position dpip0 = pipette holder module :motor pcsition start dpipl = pipette holder module motor position pipette on pclamp = position of pipette c:an~.p/loader encoder pclamp = 0 pipette not clamped (loading position) pclamp = 1 pipette clamped ~recordin~ position) rotclamp = position of rotor. sta_~e mounting for pipette loac.er rotclamp = 0 recording position rotclamp = 1 pipette filling position pipf:l = pipette filler position pipfil = 0 pre-/post-fill position pipfil = 1 fill position pipsyringe = pipette filler driven syringe position pipsvringe = dv driven svrin=a movz";ent recuired to fili pipette ! rre-set value';
cclamp = position or: capillar<~ clame/Ioader encoder cclamp = 0 capillary not clamped (loading position) cclamp = 1 capiilarv cla_rnped (recording position) pload = pipette loader empty signal pload = 0 pipettes in loader-pload = 1 pipette loader empty cload = capillary loader emctv signal cload = 0 capillaries in loader cload = 1 cat;illarv loader em~tv_ ceIldiph = horizonta 1 translator for ce;l dip celldiph = 0 cell storage encode: posi:ion (pre-set}
celldiph = 1 dip encoder position (ere-set'.
celldipv = verticle ~anslator for cell dip celldipv = 0 pre-/post- dip encoder pcsition (pre-set) celldipv = 1 capillary dip encoder position (ere-set) tdelay = variable delay beoveen clarnDing ca_oillary and startinJ to patch clamp tit = time interval dtl = pre-set waiting time interval suction oft (s) tit? = pre-set suction time interval (s) dt3 = pre-set suction time inter; aI (sl dt~ = pre-set suction time interval (si dt~ = pre-set suction time interval (sl = suction increment factor f = frequency of seal test pulse detectmin = 0 -ve capacitance ~ansient i :noise. threshold} nc;t detected detectmin = 1 -ve cat;acitanc° t.ansient (~Inoise threshold} detected detectmax = 0 -ve canacitanc° transient ~~Ineise thr~s;.eid) not detected detectma.~c = 1 -eve capacitanc° transieat~,~:~:oise thresheidi cetected SUBSTITUTE SHEET (RULE 26) . _ ~~'~i101~ Cei::lCCe i;at, I = 0 ~oC W~C!e CC:_ I = 1 W«eie ~°_:l :::cde e~taci:s~.e ~i= ~;e~:~~~ _ Dre-set ',oita~? :es; DulSe Cl::i = Cu::eP; .'°CCC:.ed DeC',Veen DCe-SeC C~:rSOCS CLI:.~.~
VOICaQ? SCC~
tCSCC:::: = L~C~-SeC Va!ue :OC C;:Cient ie.~.Cll:CQ CO Staa''t e;C a ~ l, C :''i.T.e Cal DCOCCCO
''aiVe 1 - ~ = SOIL~C:C: Va.' / S CCn I!~~c t C t0 CerL,1 i0 :rC . ._ SLDpIV Or SOIL !OCl ~ ' S C1 CISC!
tv = ame rote~rai or 'nai~.'e ac tiVat:on i;~ r°-set; .
~r~.'.~. Va:'!e = CC:ICrC!S SilC::C~ .~CC'':' iC CC:=t~cjOn CISa.
Ccr.~ro~ Lcg~c - Seco~c ~'.-:i~oc~me :t CC= __.._ _ J .. "..,.._ __ J .. r ~ _ ~_ '__~?" C,~,'.LC~~',l2 C 15 _--'.~5~~ ;,.'_. GS a __C'ri C: J WTU .__ _ -,L:'_~2 ~ . :j::°; W-~~:!
=n~wr .__ ~r_a ~ ~ ~t,~
C'-~.CidLll d~°_ v,.._ CL:~ t7°W;'::.
UO InlClailSa:_C!':
:=~.0 CCIr = ~D1~U
DC:a:_'1D = 0 ~DI!1 = U
C VtC:~'.:.~~n = 0 CCIaI_:~ = 0 C°11~:~C1 = 0 daDD = daDpO
RtoC >~_ =.~y1 i:i!on = inoise D = 0 I. Gload = C and cioad = O teen GG T O a 1 If pioad = 1 t:':en ;e~ou. "Re-lead dinette caSSette' and GOTC i9 I. cloac = _ :e.~. :e~c~ "Re-~oa~: ca~iiarr c:aSe~te" a~:d GO T O ' 9 _ . !
0 ! .-~utc''°d Move ;.ac~lla.~.' ~;:ar:~.~ motor c.c:am~ = 1 ~lp~/~ 'v,ei y .(~.:C~ t~ai:S;a:CC t0 ~.e~iC:CC
~i0';'2 ';.°!;Clp'.' t...=:SiatOC :0 CW:'ir'V =.
t ' _ ~~IO / ~.e:=Q_~';' ~.r.S:;::CC tC Ce!!C:D'/=;~
~~C ,,~Il ':
VIo~ ~.. ~:~-: ~ara!ator to : °:ldiDi: = 0 St;~ t:r:':e- .
'.~,a:t t-c' 'a.~ai,:e 'e:". _ .~e:a_V
G~TC ~~o 0- P:De;:e !o~d;'~:i SUBSTITUTE SHEET (RULE 26) W O 00/34 i 76 PCT/G B99/040 i 3 Move pipette clafilp motor pclamD = 1 Vlove rotation stake 180 degrees rotclarnp = 1 move pipfil until pipfil =
move motor driven syringe drive until pipsvrinQe = dv move pipfil until pipr!1 = 0 move rotation stage 180 degrees rotclamp = 0 move dt7lD = dDlD 1 03 Junction null Seal test sicr_al on Compensate CLast Move natc:: module down until Imon >/< Incise Record patch module motor pcsition d=dl Activate Junction null Measure Rtot Rtct=Rpip Ii Rpip <IONI and >/=~'_M GCTO 04 else Repcrt "Pipette resistance out ci ra~.~e" and GC'T_'O ~0 04 Formation o. Gsea1 Measure Rtet Ii Rtot=/> 2Rpip Suction or. P=-pmmHc ,; Meve patch module down until d=d2 (d2 =nre-set reccrdi~~ position) GC';'C O S
04.1 II Rtot <% RDi Wait icr t-me tl Rite= time tl mcve patch mcdule upwards r ~ ~.-L~1"._ RtOC>ZRD1~ t_'~an. St~~. vVa':... '~i_lu.'_1 SUBSTfTUTE SHEET' (RULE 26) _ _ _ ______ mc-:-~ ~~__ mcd~u=~ ~c. _, _ _ _~c__ __ - c~:.r~
~_.~ mc-~e ~a~c:~ ,-nc~c= moCU~.e dc~,v_~_ ur_~i~
C=C , 2'_~_ C-~~'~~ C'-'_ . _ L_='_~1~ -: ,~L 5 ~T,c_.~.~,:L:,~,, ____cCl"__ , ___~__ '~vm':~
.,.___ _~__ __ 1~ 0~ . 1. a1=~i--SUBSTITUTE SHEE i (BULE 26) Measure Rtot with -~.mi~FiQ for tir.~.e inte:~W delta t?
If Rtot >/= R I and dt <,~= dt?
T hen suc:ion off P = PO until Rtot =i> R? o; dt=tl I: Rtot < R I and dt >t 1 Then repeat 0~.1 until ~_~ or Rtot =;> R'_' I~ _ ~ and Rtot < R? Then suction on -~p:nm.~:a Repeat 0:.1 until N = ~ or Rto; _/> R'_' It ~i = ~ and Rcot < R~ Then suction on -xnm..nZ:.~Ia Repeat O~.luntil ~ _ ~ or Rto~: _;> R= or s = pma,;
Ii .;=pma~ and.Rtot < R? Then Report "Lnable to obtain G
seal" GOTO?0 IRtot =i> R'_' then GOTO 06 =or whole Cei1 Mode or GOTO O8 =or Cell :attached Patch Mode 06 Whole Ceil - Thresho:d VLethod Compensate Cfast I=0 hn = vhold 06. I Suction on -~mm.Ha until I=1 or dt= dt~
06.-Transient detection I~ _ ~i _ I
Ii detectma~= I and detect:nin = 1 Then I = 1 Repeat 06.? until detectmax = 0 and detect~-nin = 0 or V=
~0.
If ~ = 10 Then GOTO 07 Ii de:c;:~:aa = 0 and detectmin -= 0 -hea GO.'0 06.1 07 Cell quality test ~:easure Rs and Ihold If Rs --~> ~Rpir Suction on -p-rm~IQ and start timer SUBSTITUTE SHEET (RULE 26) WO 00/34776 PCTIGB99/0407~
V~~h~a Rs < ~R~i~' sucticn of Iv I::old < ~ '. ::':en and Rs < R~i~ t':~len see! CesC si=-ai c'' ;:r:d GO~'0 C
.in0ld >i_,7.-'. ~~d C:r:IO in~ = dC= LI'lOn GG _ G -r'~
IRs =;> =R~i~ a::d time ~r _ ~' - C .~
- :~tJ :'1~.~ sUCllOn on r. -~ L; a rl:_:
._ RS < ~t~-W!~ ~.,C 1:":C'iC < !p:i C::~;: sCal CO$: si~i:~l 0 r . I. 4::Q
GGi'~ G~
OQ E.»~:imer.t~: ~roocc!
Cl~:~ s. ''~° vcC_.'~ Jtep io siri~::?V
~I~as;~re ~.~,;--e;:t ~~:.-::~ durr~ vot~~e ste0;
1. v.lrr < :'..'Si~l_~:'. Cli~~ sii7~ ~'~iC.~T° j ~'~°DO!'. ~~CC;iC:~.l C;::'.~,~,C G~.iC GI :;ula~
I ~ i=
a ;.:~: ~ _; > Cest:.::r.- ::..;:woe ', o! Cage step arotocol GGTO G
Mle:avr° Rs T: R~ _,> ~_=;,;_ _.~~ ~;;~It~' nr~to~oi and GGTo c-~I~CSi~r° 1::'O;C 0'.:r! C' r'v0! C)~t~,L'0~.:~ VOlC~a~ Ste :PCe ~S
a v .~ Ir~oa Q 4 ; ., _. Ihol~: > io:~ :!:en stop vo..,.'~ ste~_ /drt:_ ppl.c,.Ci~r.
eroCccol and GGTG Ol 09 Voltas° step protocol Uses pro°'.lf:'. ~I:COC:'~ W'CIiCDI~
. . - ~__. :_':LaC :.01_ Qr'll° 0~~11COCOU sllD-rOLtlr!e Ill 1G Dr'.:~!C0:'.IpGli:u_. ,i~~ilC::C:C:':
V~:' ~ C;;
_ ._. ;=.1_ ::, D!1C::C:Ci: u.:i..i:uC': ''.~'..:Ve! Oi: ICr Ci!ilC' lI:C,~"'.','_.i CV
SUBSTITUTE SHEET (RULE 26) - s-Viove drTaQ application -nicrornaniculator down to dl -dape'~ ~~B -ve value represents downward movement) Move dapp = d1 + chop? and d= d? - dapp~
SlInulLane0uSlV
Move ~ = ri~ Qlass caDillarv moved up Call software controilin~ flow control valves 1~ Reset .~.utopatc:~
GOTO 00.
?0 Steps 15 or_~Nards are routines for setting up the apparatus controlled b~r the seftware/methcd a_nd do r-of relate to the inventive operation of the apparatus and their design is within the normal competency of the skilled person. Steps 1° ar_d 20 relate res~>ectv~velv tc reloading t=.e n~.Dette and capi l.larv cassettes arid to checking and/or reset:tin g of the apparatus if operation is unsuccessful.
Figure 9 is a flow diacram illustrating steps 03 ar_d 0~'_, which relate tc formation of the G-seal. These steps comprise the most important advantageous steps in this embodiment for centre=',ira t=-!e met~.od and apparatus is described herein.
In step 03 (junction. r.ul l ? , the pipette tin, is initiall_~
spaced below t_~e men,_scus at the end ef the capiliar'_r.
'T'he lGQiC, Cr S~;itwcr°, t~_~_en COntr.~,~ S tv'"-a pa~Ch mCd':'1°
mOtOr t0 mCVe the ~~peLte t1p tO~.Jar~S the menlSCllS unt'-~
20 C:WtaCt .S maC:., Cet°Cte.~. bV e'~eCtr'_C.l C~ntaCt therebetWeen. '=~'e mG-v'emeTlt 1S then S~.ODped W_~_1°_ Che SUBSTITUTE SHEET (RULE 26) WO 00/3.17 r 6 PC i /G B99/0407~
J1~ette reSiStanCe =S measurer' ar_C tue _ m~.t.._ ''CS'itlCn reCCrGeO ~~" ~ c -- , a.. shcwr_ i n _ .;ur es - r ~
lb a__~ ..! . __ Rtct is outside a precete- n ,.
_~:i e~ =ane~, t he e~per,~ment ~~ aborted.
iil Step Q=, RtCt ..S medSUreQ arid 1_ 1 t =S aDOVe a pr°Cetera~lned t:':ieS:O_r', .t ~S assumed ti_at a cell iS
pOSltlCned C_'1 ti:e DiDette tip SC S1=CtlCn 1 S appl'_2d tC the plDette anC t~"':e lOglC CCntrOl S t .e pdtC _~ mOdlll a mOtOr t0 move the pipette _ fur~her ~n o t.. l _ - t ~~~:v_d witi-_~ n the capillary t~ a ~~edete~~:_r:ed recer _.~ .J position (d=d2, as ro sh C:v-n in t --~.:res =~. ar_ - . ___ 1CC; C t__~__ m0'feS ~~ SL~.~, 05 t0 test t_:a G-Sea ~ .
Tf at ti:e Start O~ Step Os, RtCt _~ 1eS
s than the DreQc~''er.ill neC<' t~r°S.:C_C, the _.~.Q'~C assumes t._at there iS
n0 Cel 1 at t_'1e D;-~~,ette L1_"~, . '~'i1e 1 CG1C tu'=e D Wa'_tS LOr a s predetermined tire ir_ter-.al __ be=ore control _ 1 i~:q the patch module motor to move the capillary away from the pipette u:ltl~ RtCt 1S measured t0 ~:: 'Tr.Ca er ~- ~,- t tan the preCletermlr:eC1 t:'=reS~'?Oi , 'nii:e_~: L_'=C~ ;,~,CVeme':t '1S StCD_ DeC.
It is believed that ~__ t::is positio t=~e pipette tip i s stil 1 zo in cor_tact w;_th the '.~ic~,:y ;__ the capiliarv but er:lv V"_a a neck, or bri dge, ef 1; ~w_d crasar: cut '~_~ surface tensie~.
between the c=pilla a~~
_ ry __ the pvpetto. The logic then waits until Rtot drops tc equal the predetermined threshold. The logic t'_her cCntrcls the patch module motor 2~ to return tha pipette tip tJ d=d'_, tie position when it first cor_tacted t:ze capW_~r_~ meniscus i n step 03 . lr Rtot is ther_ greater t:~a_~. the - codetermined threshold i s assumed that contact wit:~: a cel,~ has beer. made at the pipette tip, suct~...._ ~S aDDlleC t., t_~e p1 Dette ~" t_r._a al:~
30 log"iC CC:aro~S tna DatC:~_ 'T1CC';:le '':CLCr t0 t.,CVe the p_ lpeLL°
lnt0 t.'=e Cay~,=.__~rV L,r t :C i'°Qeter~11: ed reCCY'Cj._~_C
DoS';t10:1 a t d=a2 .
SUBSTITITTE Sg~~T (RULE 26) WO 00/34776 PCT/GB99/040'r3 It is believed that waiting for the time inter~.ral t1 which may be betweer_ 0.5 and 10 seconds, cr preferably about 1 to 5 seconds, permits movement cf the cells at the capillary tip, which is encouraged by the mcvement of the s pipette tip to draw out t:ne capillary meniscus.
If Rtot is still less than the predetermined threshold, the steps of waiting for time t1 and slightly moving the pipette are repeated for a predetermined number of iterations until a failure cor_diticn (step 20 ) is io reached.
Control Logic - Third ~mbociiment Figures 10 to to are flow charts illustrating a third embodiment of the cortro,~. logic of tt:e invention. Aspects of the third embodiment are, where appropriate, ccmmon to i5 the first and second embodiments. The third embodiment, however, incorporates certain improvemer_ts resulting from experiments by the _nventor.
Figure 10 is a flow chart showing all of the operations of the ccntrol lcg,_c, or software. This is termed the zo AutoPatch system. Figures 11 to 16, and figures 10a, 10b, lla, 16a ar_d lob, are e:cpanded flow charts for ecerations within the flew chart ef Figure 10.
Figure 10 describes the setting up of the AutoPatch system, irlc~luding the initialisation of all the relevant zs hardware. This involves the steps up to starting the test sweep 302, after which the steps of interface or cell location 30s, Giga seal testing 306, whole cell detection 308, qualification 310 and experiment: 312 are performed as described herei-_. Note that during the patching process, zo t::e movements c_ the capillary and the petri dish are locked together by t:ne software in order to maintain their SUBSTITUTE SHEET (RULE 26) posit=ons r?'~atiVe t.. eac~ c-: er .. _ _ ..nS to>._ . _":e ;r;Ovle-.,~.n t CZ the er~_ p _ _ ..~SL: :_aS nC rC! a 1~ . CCeSS .
_~ DatCn~ n G ~~
Ti,lLla! _:' t ne C3p~.~_a='J a=:Q ~etY'~ di Si:_ ;llCVe tC'niarCS C,'?e p;.pette dt a raD'~G SCee~ to a Dre-Set diSCa~lt (Step s in order to pcsit,~cr_ t_.e lic'~id,!a-_Y intern ~ r_n;, _ _ac~ wv._ __ aDDrOYliT',ate~V Z;T,IT; Cr t ne :':.uc''tte t=D. "':ilS ini tid~
(roug::) posit'-on ' =orm d 1 t per_ a __ order c shorten t a time inter Va'_ 'etWeer: staY,.__ ~ ~ ~,g __ pat~___ ~.rocess ar_d reaching tie ._ -=°r=ace. __e distance between the patch ipette _ a no ~~-:e i -ar_ _ _ _ _ , ___ ___~ __~~_ is ______a~i-, '_araer t .a n ?~;~.:;: in order tc a',icw s~':_~icient space nor loading o= _i:e p~ pette a nd t_te c ~ ~ ~ any. a=teY
_ _ _ _ cu~h positioning, t~:e seal test pulse =~ started (step 3'_~) and the trd'_SldtOrS ICaD~.i~aY:i anC p°trl .~.'_S:''i are SW~tCneC t0 __ S~ cW SpeeC p"'=..~ t0 2 ntr: CL' t :e Dlpette i ..,~,. A
_ :_~~ th..
t.. L
1 ,_~~~G/ din lnL~-_aCe.
n t' lgureS iva an : ~'v~ a ~'DanC the Sale Cli'rt rCUti ~_e il ~ GnC
L~ a reset r:Cut~__~ ~~,, =_d '.~s._ at Var-CuS pC~T-!tS i:'J t_>_ corarol logic cr sc~tware.
2 C h ; Q a r a ~ _ 1 8 a L ,~ O'rJ d '~ 3 C a ~ ,., ; ~ r- ~-, _ram a~a_....___~.. t__e '_~ter=aCe O'"
Ce'11 lOCator rC;a:. n~ ~~~Ct= r-figure 1',~,. lI: t..'_S
rCL:t~n°, suct;~or> is applied to the ~__teror oz the pipette, and the Cap'~~ ~ 3rV a:':d p etri di S:: are ;VOVed tCWardS the plDette at a slow rate (e.g. ~~~ micrcmeters/s) (step 3?2;. The 2.5 C~:rre?"?t iS CfICn~CCre.'". aLCer each Sweep t0 determi ne '.J.~e.:
the inter'ace is detected ;step 32,'~ Tie terYa :, . in _ ce ~s deteCteC bV ai: CL ?'Set ~n t_'!e baSel i:-,° Cur'"°_Tut Cr ti?e apDear3nCe O' ._ ~,.>_re_~:t .~.u'.~Se 'niClen t_Ce C ~ is _ _ r w..'~ t ~ made .~t~.C . _i: CO__~:traSt ',J;C__ CO:1'.;entlCnai DaCCh Clamp, Sea=
30 LOri'laticn '.vit:= Cr'_e ,__~e?'=a_._ =atC:: Clamp teCh~ ~ C=~
__iQ:.e ,..__ CCCur V~YL'.-a_ _ slm:»..aneCl=S_' ~~.. r-C
w-~ ~. ntaCt }'JetWee?': C>~a piDett°_ _ ar':~ t__' el:teY_~_3_ v3C_~_::~ SOlL:tlCi: at t:":
suss'rrrz,~ s~ET ~m.E zs>

inter=ace 32b. The logic distinguishes between an open circuit (pipette tip i_. air? and a rapidly formed seal by menitorirg the current trace for the appearance of a capacitance transie.~.t at th a end cf ttze seal test pulse _ (step 326). This transient is due to the pipette capacitance, which increases as the pipette is immersed into the solution at the ir_terface. As the pipette tip touches or crosses the air/ii~uid interface the caaacitance is likely to be undetectable amoncst le electrical r_oise. As the pipette tip moves irao the lieruid, however, the capacitance increases until it is detectabla over the noise.
After the pipette has entered the interface, the ca~illarv (and movement cf the petri dishy is stopped, a junction is null may be performed and the pipette resistance is mcnitored. The presence of a cell on t:ne pipette tip is indicated by an increase in pipette resistance which must reach a pre-set value before the iegic will allow the system to proceed to the next step. After a cel'_ has been 2c detected the Giga seal test 306 is undertake.
Figure 11a expands the interface hit i:outine 330 of the interface or cell location routine 304 of Figure 11.
Figure 12 is a flow diagram of the Giga seal test routine 305 carried out after contact with a ;:e11 has been 2s detected in the interlace or cell locaticn routine 304.
The Giga seal test routine 306 comprises a repetitive loop 332 in which the level of suction applied to the pipette interior is increased in pre-set increments 33~-- and times 336 while rncnitorir_c the change in pi:cette resistance.
3o The suctior_ is ~:ncreased until tine magi mum vacuum i s cbtained er Gica sea? ~or<i~aticn occurs . T!-:e lees ends and the sucticr: ,-s switched o'f 338 i= either cf these suBSTrruTE sxEET ~~ 2 s~

WO 00/3:1776 PCT/GB99/04073 co ~itions __ satis__ed. ~w,e _ _ _ a:~~.:~u;~ s uc t'ior_ ha s :~een appl led Lit ~, Gig sea h _ as :~~~ -caned t:=a 1 oco r~GcatcC u;:t=_ (=~.~ga sea _.~ '-'tl Cr r:,:~ c_~. _ time-cut . ~'he LCrmatl0n C= a ':'~:c Or_m Sc~'a_ ~S renu'~re~ 1:1 OrCe'" t,'C allCNi S prOgressiOn tC t_ie : eYt Sty.-~a.
Figure 13 expands the w'_o~_e cell detection routine 3C8 cf Figure ~.C. T_n this rc~,.___ , the transierts ,~; w d a to 'ne pipette capacitance ~_~ cancelled - 1-anc this is =o _cwec b'_~
a repet'~t_.Ve lCOp s .O ir: 'n%:.1C_~_ t .e ~ ~'Ie~.. cf sllCtlOr 1~ ai:Di~red tG t::e ~ :et'e ' -er_ _ ____ __.._ '~S ___.,reaSeC ._. ~ ..et srCreme~tS ~ .. ~ tlmeS ~ -,.
- .- _ a_.c _ _ vahi l a mc..~ for ing t he c,~-rent t..e _ _ __~__ oL capaci Lance transier_ts .
j'~C f GY n a~V°="' ~'e ese tra iS;.e_TtS are d',1e tC t_'?° C_"_arge anC Q1SC_'large CS t~'le Cel ~.
membrane ca c a c - ,.a_ ..o o' t pa .t n .. a_nc are __di~ ~_. ~ he fcrmaticn s of whole ce_ =ecor d_ :~, mcde .
Figur a '~,, ex: and 'he y , S ~ Cl:a! ~=_ , -,~
_ aW. ...~W.. 1 of FlCure 10. ~n order to ~:ali=.~~ _.._ wse ___ an e::per;_ment theycell must ex:;:i~it a -,cl tac~ aate~ is G--hew _ _ currer_t e~:al to or greater ~_.~._ .. ore_deter-~ir_ed a:;.'_itude a_nd ociari tv ?a 3~8 ~__ res~cnse _.. a test poise. ,-- .
~ua_~~icaticn proceeds u=~'_~~-~ ti=a Ce._~ ...__==eS Cr _ t ~.;;le0ut '~S f'1~ t . ~ur~ nC
goal i=icati o_~. t he ~:a'.~-it-, mcnvto_ _ r 3 ~ run also.
g _.. w Y,~Cw oiagram c_ the , ~, r. r Fi ore 15 rn..a_~.ty mo._i,.or rcuti-!e J5C of F=cure __ a_.d ; , c;escribed be~ow. ~ t cCmpr,ises a 2s repetiti-re =ocp v~n which ~.. _ petty suction is varied response to measurements of series resistance (RSeries) and currera ;~~lon) . Currer~ , _ - y_~~r trough t.e cell membrane -~'_a the -~ oette --~ner _ _ =.tes a -reltage error due RSeries . _:~:e va__~ ~_ _ _ _ _ ~r.CreaSeS Cur ~nQ
~e whole ce~.l _ecor~_ri and t__is e_fect ..an !~e reduced bw the applicatio : cf _~~~_~__ _~ t h' pine t_ t ~ inters. _ ~5~. :.~, _ ;crease ~._ t:~e _.__ ~~rr~r_~
__~_ ~_ a - t'_ne '_hol ding SITBSTTT'U~ SHEET (RULE 26) WO 00/34?76 PCTlG899/04073 _ ;, _ potential (usually - 'o0m~~) indicates loss of the Giaa sea'_ and this car_ be caused by excessi~fe suction. Acceptable values for RSeri es a__~. I~Icn are entered in the settings for the software. The quality monitor runs bcth durir_~
the qualification stage ar~d the experiment.
Figure 16 expar:ds the experiment routine 312 or Figure 10.
In the experiment routine, movements ef the petri dish and capillary required ~o carr-Y~ Out drug appl ication b~.i t.~:e method shown in figures ~-7 inclusive are carried out.
io poring these movements the dish is filled with e:cter~_al solution/exter_nal scluticr_ plus drug via soiencid operated flow control values 33b. Sefore drug is added, the current evoked by tr:e test poise (or pulses) must be reproducible within a pre-determined percentage (entered m in the settings).
Figures 1o'a ar:d 1'ob a xpand the trace ru_~. routine 334 and the stabilise routir_e 350' c' the e::periment routine 312.
SUBSTTTLTTE SHEET (RULE 26) WO 00/34776 PCTiGB9o!04073 Figure 17 s:_o~~rs r ecorci~cs cf c ~_ _ ____ ( 1 ' ~ c1 rage ( 2 ) a nd ODtalne~a L_Cm aa'.ltC,ilale.., h ,-l CatC ~._ _ S_~'SteT,Tl ~,.-.l~tC~aC_: ;
ShOWIn the L.. .l. CL ~"' '"..
=g ~'r,T~a~'1 _ ... J Ceal ._.__~...._ SoLtWare CCntr01 uSl r J t_'!e _ =te=LaC°_ ; 3tC n ': 1 a,~' ~,D t e..!'Y_ _ ' .
RECCr~l:'_gS frcm d.~_ V!rii Ce~._ .
Figure 18 shows recordings of current ;1,' ar.c voltage (??
from aautomated interface n -- ?~ ' ' . _ ) a~~c damn system (:,~~to~atc'~
showing t:~e ir_crease :._ ~ nee ~a:lca__ ~_ar_sier:t cbser-red after moving t~.e capi i~_ary t., posl~io (- -d ~r_ d~ reccr s a and ~o b) . fc) and (d) were obt~ : ed =.. -i c _ ~,_~~_ ~~~oma~
compensation =or pipette capacit~__'_ ar_d a c?-:ange .__ the 1101 C=ng pote::t'~al t0 -~'.Qm~. 'eCCr _'.. g Obta'~r.e~ from C___ same cell as Figure 1 ' .
igur a 1 ° shows reccrd_r_as . _ _.._ ~ ent ( 1 ) and -~-01 rage ( ~ ' is from ar~ automata interface pate damn system (e~utCPa'tCi?J . ReCOrCS ~a; aC ~.~) 'mere obCalneC after v-.lCa seal formation (ce'~= attac':ed hatch mode) usir_~ t_ interface v.~.atCi Clamn ~eC i_~_ ,y:e. i_~.e a~Dl lCat_GO c'i SllCtiO n t0 to ter_.._ p'_pet to ~__ uncer f ull scf aware 2o contro~~ ruptured the ;~e.~.li:ran a patc'~ to obtai n a o =
t h ,,,r'.~ 1 _ ce 1 1 recent=__~_gs so~~m i~: ' a c, _nd " . The establ _..hme~~
of the whole cell :~cce ecc s_~own by the cf _ .ding i s presence ef the farce capac~tarce transients or. the cur=ent trace. R c r. ~~ o' ~ L
a o~c~__J ntaire _cm the same cell as 25 f i gllreS 1 % d_nC 18 .
Figures 20 si-:ows reccr~in cs c= me.;.:crane current (1 ) an d voltage !2) ir: whole cal'_ reycrwln g -rode obtair_e~? usi~:~ a_ autOmatea __ vlamt S
patC VSC°.TTl ;~llt.~.__._Cnl 'nllllC~ emD_C'.'=
inter Lace GatCi. CIaiTID teC n_~_ _ _ . ___~ riCl C_. J DCte:utl_._ 30 WaS -o0T'1',i a: d (~; ...__~. (.~, SuC:~! C'~;tWay ~ DCtaSSl'~lm C:i.r_..__CS
(ICvI .1; ___ _eS~~C::S° tC a y"_ ge SCe,, tC +J~m~~. aeCCr :__ -, O.~t...~l.... ro,T, _. Same Ce__ an d .
i-~ ~i-t.~~ f tn' ~5 L'._gi~reS _ i , i~ l O
SUBSTITUTE SgEET (RULE 26) Figure 2i shows t'ne e==act of the pctassi~~m channel "
blocking drug tetraethyiammonium (TEA) or. the potassium current reccrded tram MK1 cell in whole cell recording mode obtained using the Interlace Patch Clamp tecrw.'~u°.
s After the establishment of whole cell recording made the cell was positioned in a recording dish by the method shown in figures 4 to 7 and described in the text.
(a) shows the current obtained in normal extracellular solution io (b) shows the effect cf replacing the soluticr: in the dish with eYtracellul ar solut~.cr_ containi nc; TEA ( 5mM) .
(c) the blacking effect cf TEA was reversed by washing.
It will be read,_lv appreciated by those sl~:illed in the art that.
~5 1. The stability oz recording using the interface patch clamp technicrae may be superior r-o that of conventional patch clamping. The greater stability of interface patch clamping is bf>cause the ~~ell is held by the patch pipette alone. In conventional 2o patch clamp recordings the cell :is held by the path pipette and a solid substrate and vibration tends to move the pipette relative to the substrate causing loss cf the G-seal. The interfaca patch clamp is, in contrast to conventional patch c:Lamp apparatus, 2s relatively insensitive to vibration durir_g drug application.
2. This method of drug application could be applied to a plurality ef recording pipettes/capillaries and form the basis for a high throughput electrophvsiological 3o assay system. It wv~.l1 readily be appreciated that the Interface Patch Clamp techni«~e could be used with multiple pipettes and multiple capillaries in a SUBSTITUTE SHEET (RULE 26) WO 00/3x776 PCT/GB99/0407.i mG~ ne ~ .___ ~lJr:l.'_', ~ ._.....__ ;,iette ~~-,,~,..rS -_.
_ _ __ DeCtlv'e a_i--~e~ c ~ ~_ - .-,d_ _ ; d.. , , ..
_ - _._._.__~_ ___ _ . _ .~Cyy _ ___ _~r'~,~e__ a-~ CCC°~~.ar . .._~__,. J _ nCt ~......__ent_'r " ~__.~rer ....
_ _ .., S1=1~C,;e yL"_~?~ Le ._,"~.~ ., ~~~,e '_:S°C SV~1C : __. Ca'.:Sed tJ
O._.~.~_ more tha:: O~:° CaD ._,., e'L:c~':ltld! 'z ;
_ _ s _ ~. ~Iu, t ple ~atc ciamz~ record~_naS c..~,_._. be ~;,ade ei t:er Se~~e__tialla_, S-mu'_tan ecusl_n, ce~~nc--_~_~ on the a~~i ica~_on.
-,s ~Nas mer:tic_~.ec a~cV, _~ __ .~." esser:t,~a_ -c t:-~= gerera~
pri ~ci~'_e c_ t_=a _n-re~_,~cr< ~J use a c,p-_~~lar-,; in c=er to Great°_ a cc~~u:~~ c_ - _ _~ ,,______ _l~r-es rise t~ a =vcuicj'a-=
___ter=ace at w~;c'~ C~__s c___ ~e ~'_CCatec. Ct:~_er Wa-!s Cap a 2nVlSaCeC 1W:=_Ch '__.. Same e~=.. _.~ Cai'! ~..'Je .--'.,.~..'1'_eVeO.
Fcr e:c,~mpl~, ~_ shc~N-n ~__ _ J __ 22, a drc~e~ cr 'lcb" cf 1 = 1 ~.'C'l.:-.~., ma;i C"O ~e C
CrCV~._'" O_I G 5~.;r;vCr~ SuriaCe. i'?e Sur'aCe has a hcle t ;reach _~ anc t__.. d=c_et co~.rers the ho i a .
Surface terac.~_ c e~re= .._ _~_e ,~_ _ id from the droplet dl'~CC~'~._~_, ~_~_rCllC~ C_'10 _hC=. W'_t~.~L-r: ~:=a dLCD'10_ C°~.5 are S';SDerCeC . T'f? 1 S ~ 1 r, a_ C VS cCCeSS CC t:_°_ GrCD_et ~='ld t~'?e 2G Ce_~ S C: n taln °_'~_":er°___. , a SL::~ta~~e e~eCtrOGe SuC
h aS ...
DatCl? vl:ett". ___ ~~e arr nCJ.,ement ShO:JIl 1:1 1 CL:re ~~, meallS are ~r:~'~1.~_eC 'Cr =_~',v C~ Ot!ler il~.-1dS '~.?1 t0 anC Cut C1 ._ d;S-; .J_ ~.~__~_ _..___.._____ .._ 'rJ!'~'_Ch t..°_ Sia:,OCrt SurfaC°
tail t t:=a ~C~._~? lr: ' ' _,~_:,iS a 'n%a1_ . hnCe a C°_~ ::aS beer a 1't a rr 'r'~ o I a n c V, ~-~ o r 1 i ,_m, ~ i c o att~c__..~ to t__C ctr"c~, t__'_ c.. may b_ introduced lr:tC t_Oe COr:ra'~ =~_ eltuc~ ___ CatC:_ mCG° Cr lri f~ OW-~'.r',rCL:CI1 mCde 1.~. Order tC reSu~_~ _._ t_'=e Ce__ ~eI::CJ eY~OSed 3t 1tS
i Si_riaC°_ ~_. tC_e S'.:r_CL~CI~~ ~ lu .',1C. 0112 r~ V '~Il SL1C!~ aP arr arlCe!,~,ent , ti!C Cr 1 J 1='1a~ 11'u'Lil~ a.~_G tPe ?"ema' :_ =CJ
un-cttaC_~O~ C°~15 W=~ ' ' c z f_r t'"e - tQ :C- ~O be WaSWeG~ anial O~.i; __ 3G area ~_ ~_=a e_e_~rCQc, _~2.
SUBSTITUTE SHED f' (RULE 26) WO 0013477b PCTIGB99/04073 Tt .S W=t.~'1=_? t n°_ SCOpe O- tile ~.n'Ie_'!t.;::1 t-"_at G=O"
etS
~I'.rC~=t be iJr : V =de_~_ Cn i_,~i,-~erLC r?.te S'a=~IJCr t 5i:;~=aCeS .
_.., shcs~r! i:~ fic'a=a 2~, t''_e effect cf surface tension may be to a'low d-cplets of a suitable licui;? to adhere automatically to the a~:derside cf a suitable suDpert s'arface. The suoDOrr_ 'Y~ ' _ _ su_iace m=g ht ~ or e:cam~le be a CC;er Sllp OL glaSS Cr OtI:O=" ;ulaterla~. L:=C~7letS i:: '..Ih 1Cn Ce~'~1S
dr2 SLlSl~enCeC prOVide t_~e a~.r/1~~,-= : _Lnter=ace aCC~rQ~. nC
tC t~'le lnVeT:C_ :._ a nC CCT'_SeQLe_ntl 'i TIaV pe LSe~ =.__ a !TleCnCC
Of lnterfaCe r.atC~'1 C~ am~=ng aS CeSCrlbed abCVe.
~.j haS a-rc''aC~: ~J'ee_': merit-Oned, ...he a=~~anClE'!Tle_~_C Si:Cov_'!
Fl~llre 23 aS Sve! ~ aS tn a a=ra:'Cement St?O':Jn ~__ _ '_Q'ara ~-'_ ..
a_LOi~JS tOr t.~_e i.Ori'lat=Cn C~ a matrla O= Ce! i SllSDenSICP_S
SO that mLlt_~! ° eleCtrCCIeS Can be mu_tl.~.~ eYeC t0 taite readlCS elt:?ar Slml~~! ta:leOLS_'J Or SeG~.lentlall'i (aS 'rlel 1 aS
S-:1C1 V) _t w=11 De aDDreC~.aLeC1 DV tnCS2 S~:_~ ! E'_d '_n the art that a n n ' ' "D i- -r~~~ C 1 r' ~'Je reD l aced DV a co_:~e_.t~ona_ c! ass atc_. piDe__.. ou~u eru'L'.';L'alen t e! eCtrCCle. '1t 1S COnS;,~.ered t0 be W=tn-n the 2~ SCCpe Oi t~''_e y'L'reSent '_n'Ientj.Cn t~'.S.t t.''le el eCtrOCe migi'?t be e1t_ner a S?~:C_e 1"eC'Cn C. a matt=:i ~_ rer';CnS Cn a S.~_e°_t OL _materi a1 (SLCu aS d S; 1 i Con Water ) WhlCn~_ =nCCr,~..OrateS a microstructure t0 SJh'_C:! a Cell Can De attaCt'!ed anG Whi Ch WOI;I C. DrOV;.Cle t}':e neCeSSar'J elect="_Cal COIL~.eCt;On . FOr ?= eYdml7le, aS ShCWn in ~1C'are ~5, m-CrC:itr';Ct'areS COi:ld be etched er_ to a s'_lic::r_ ~Na~er (e.c an o:cidised s_licen wafer) , wiWc:~ mi crostructures would be designed and adapted to be a xle to capture a ce_1 from the iic~',:id/ai r interface o. an arra~~eme:':t acccrdi nc to the preser:t ~o inVenticr_. T'':'as, t':~:e ~er_~rmance a=d advantage ef the s ~, t ,~ _'d ~ ~..e cur~~~~t ~ '~°r°rrec n r'__~ic n c _.m... ~., ___ __ _ l ; D____ SUBSTITUTE SHEET (RULE 26) WO 00/34776 PCT/GB99!04073 cenventie~a~, c'_ass oatctiaette but would i-_clude f,.:~~cticna-_' y e~:i-.ra~_e :t means .
~s has bee_~_ described b~_~_ , a drug in i icraid soluticr_ can be applied to t~.-~e ce ~ ~ ___ a r_umber of wavs . For S eXamp ~ a the dr',:C CCLlId : a app! ~e~ 'v ~a Che Caplllar' _ r ~ _ a air interface is formed in a cap'_;iar.r tube.
Al ternati<relv t:~e d-ug ca:: be anpl led by perfusion i nto a dis(as deSCribed with r~_____.ce to figures ~ to 7i.
Furthermore, perfusion cou-~c be ac:~ie~~~ed by flowing the io drua-ccn~av~~. lic,~'_a t_ ro _ ug:~ a ~___. .,_ ccn..~iner = as shown_ .__ _ ~,ure 22.
fur ter arra ngemen t for drug application i s shown i~.
Figure ?5a and 25b. _.. this case the electrode ~or example t he patc h pipettei penetrates t'::rough the lower wall of a '.vel 1 . A s'~: pe_~.s'~o:~_ of ells is ? oade,~. in to a ube as cap=_lary t previously describe:. =_ttachment cf a single cel~, tc each p pette tip follows, as described before. Or_ce ce 1 is are a_tac.~ed to the pipette tips t.~:e capill ary t'.~~es cor_tai:nin~ the remainder cf the cell s in 2o susper_sion ca:_ a removed. Subsec'aently, a drug solution (23 ) is disp eased into eac'.: well ~ F ure 25bi and patc=
clamp measur ements can t=:er: be carved out en the cell ___ t. a er:~,rironment cf t he s'.~r_ounding drug solution.
Optimisation of Patch Clamping Conditions zs '~''_:ese spilled i~; the art wi~'~l appreciate that within t general teac:_inc contained herein for the interface catc~
Cla:';lpi_nC met :CC ai:C ail Oarat'.:5, l.t ate be ~eCeSSarV t0 _ m."
Opt;mlSe Ce. ta___ CO_~_C~~_.~::5 ~ __ patch Clamp meaS '"'e °
m~ nts .
For e.camp 1 a the ccr_ce ntrat'.~ct: and caci~i ng density cf ce'_1 s 30 ~n t he Susven SiC__ ma'r _ _ ._ _.. be ~ptimised. F'~it;ter:nOre, tile C~_ ' 1S a.IC~ ~~._ SC_._~_.,:':S iila'J ~e ~e:'.I-.~.C-'r3tu=a se=1S-t=Vc SUBSTITUTE SHEET (RULE 26) and an opti mum temperature of operati ar_ may nee ~.:~ be determined. Since the lnVentiOn relies er_ the fermaticn of a liquid/air interface at which the cells are located, it may be necessary to optimise the osmolarity of the _= suspending liquid medium in order to achieve the optimum level of surface tension etc.
SUBSTITUTE SST (RULE 26)

Claims (18)

Claims

1. A method for providing a cell attached to a patch clamp electrode and having a high resistance (Giga Ohm) electrical seal between an area of the cell membrane and the electrode, which includes the steps of:
i) providing a suspension of cells in a liquid;
ii) causing the formation of a layer of cells at the interface between the air and the liquid in which the cells are suspended;
iii) bringing the patch clamp electrode into contact with the interface by moving one or both of the electrode and the interface respectively together;
iv) contacting the electrode with a cell in the cell layer at or near the interface; and v) causing attachment of the cell to the electrode.

2. A method according to claim 1 for providing a cell attached to the tip of a patch clamp pipette and having a high resistance (Giga Ohm) electrical seal between an area of the cell membrane and the tip; wherein the electrode of claim 1 is the tip of a patch clamp pipette; which method includes the steps of:
i) providing a capillary tube containing a suspension of cells in a liquid;
ii) causing the formation of a layer of cells at one end of the capillary tube at the interface between the air and the liquid in which the cells are suspended;
iii) bringing the tip of the patch clamp pipette into contact with the interface by moving one or both of the pipette and the tube respectively together along a common axis of movement;

iv) contacting the tip with a cell in the cell layer at or near the interface; and v) causing attachment of the cell to the tip.

3. A method according to claim 1 or 2 in which the liquid in which the cells are suspended is an extracellular physiological solution.

4. A method according to claim 1 or 2 in which the layer of cells is several cells deep and loosely packed.

5. A method according to claim 2 in which the layer of cells is formed by mounting the capillary tube in an essentially upright orientation and allowing the suspended cells to sediment to the downward end of the tube to collect there in a layer.

6. A method according to claim 2 in which the capillary tube is mounted essentially upright with the interface at a lower open end of the tube and the pipette is mounted essentially upright with the tip upwardly pointing.

7. A method according to claim 2 in which the capillary tube and pipette are concentrically mounted with the capillary tube in a fixed position and the pipette movable along the common axis.

8. A method according to claim 2 in which the capillary tube and pipette are concentrically mounted with the pipette in a fixed position and the capillary tube movable along the common axis.

9. A method according to claim 2 wherein gentle suction is applied to the pipette during contact with the interface and during the step of contacting the tip with a cell.

10. A method according to any of claims 2 to 9, in which contact between the pipette tip and the air/liquid interface and/or subsequent movement of the pipette tip into the liquid is detected by monitoring pipette capacitance.

11. A method according to any of claims 2 to 10, in which if no cell is contacted at or near the interface at or within a predetermined time after contact between the pipette and the interface, the pipette is withdrawn from the interface and then moved back to the interface to repeat the attempt to contact a cell.

12. An apparatus for carrying out the method of any preceding claim which is a computer controlled apparatus including the following elements:
i) a patch clamp amplifier;
ii) a source of variable suction for a patch clamp pipette under the control of the patch clamp amplifier;
iii) a holder for a capillary tube to be mounted vertically;
iv) a holder for a patch clamp pipette to be mounted vertically in the same axis as the capillary tube in an inverted orientation with the tip pointing upwardly;
v) a manipulator for controlling relative movement of a capillary tube and a pipette when each is mounted respectively in its holder, the relative movement being along a common axis of movement under feedback control from the patch clamp amplifier and allowing for the tip of the pipette to enter a downwardly facing end of the capillary tube.

13. An apparatus according to claim 12 which includes an array of a multiplicity of capillary tubes and an array of a multiplicity of pipettes.

14. An apparatus according to claim 12 or 13, comprising a pipette capacitance sensor for sensing pipette capacitance as the pipette tip contacts an air/liquid interface at the end of the capillary tube and enters the liquid in the capillary tube during operation of the apparatus.

15. A computer-program-controlled patch clamping process for carrying out the method of any of claims 1 to 11.

16. A computer-program-controlled patch clamping process for controlling the apparatus of claim 12, 13 or 14.

17. A computer-readable medium carrying a computer program for controlling a computer to implement the method any of claims 1 to 11 or to control the apparatus of claim 12, 13 or 14.

18. A method for controlling a computer by means of a computer program for implementing the method of any of claims 1 to 11.