CA2002660A1 - Method for electrical detection of a binding reaction - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A method of detecting a substance in a test sample, which substance is one of a pair of substances that undergo a specific binding reaction with each other, in-cludes the steps of reacting the test sample with a reagent comprising the other of the pair of substances and determin-ing the extent of reaction therebetween. Particles having a first one of the pair of substances bound to the surfaces thereof are contacted with a layer of the second substances which substantially spans a path between a pair of spaced-apart electrical conductors superposed on a substantially non-electrically conductive base. Each of the conductors is connected to means forming an electrical circuit which in-cludes the conductors and the path therein. The binding reaction between said first and second substances causing the particles to be bound to the bottom wall to form aggre-gates. Unbound particles are removed, and exposed outer surfaces of said aggregates are coated with an electrically conductive substance. A change in the electrical current flow through the circuit caused by formation of the coated aggregates on the path is measured. This electrical change indicates the level of the substance to be detected in the test sample.

Description

2~ 6~
~T~O~ FOIR ~ECq~ICAI, DEq!ECT3~02~ OF A BI~ EACTIC)~

AL FIELr) This invention rel~tes to a 2n~t~od ~or ~lectric~ detecting ~ binding re~ctic)n between ~ pai~
5 of ~he~ ub~tance~, particularly bi~genic s~bs~nces such a~ antig~ns and antibodi~s o~ nucl~otide~.

~IAC~RO~ OF 1~ I~IOIa ~ e~en~ly 6everal type~ of electrical immunoa~say teahnlques have b~en developed. One ~ush 10 technitaue utllizes ~ield efe~ transistor~ coated witA a l~y~r of antlbody in the ~ate re~on. I~ AD
an~igen-antibody re~tion ocrur~, the charge ~oncen~r~tion o~ the t~n~i~tor ~haslge~. Ex~mples of thi~ tYPe 0~ ~Ystem are ~iv~n in ~ahenck tJ . ~ . Patent NQ .
~,23B,757, iS~Ued ~eCember 9, 1~80; GUCke1 U.S. Patent No. 4,1~0,7~1~ is~ued December 25, 1~1; Malmro6 U.S.
~?a~ent Nos. 4,334l880, i~ued 3une 15, lg82 and ~,4~4,8~ U~ April ~4, 19~4, and Japa~e3e Patent Publi~a~ion ~ao. 60-2~6$8. In another ~ystem, a body 20 flu~ d containin~ an analyte to be detected i~ d~poslted onto ~ ~urf~cq which h~ been coated wi~h ~ reagent that ~ind~ ~peci~i~ally to the an~ly~, 60 tha" a bindlng reac~ion take~ plac~ . A tagged re . gent i~ then ~dded which r~act~ with the analyto-reag~r~t ~omplex or w~th the 25 reagent to ~hang~ ~he ~lectric~l reactan~e of the ~urface, See Eber301e U.S. Pzl~en~ No. 4,~19,~35, ~sued ~ug~t 26, 1980.
Save~al oth~ me~hod~ have be~n proposed for mea~uring inununologic reactiona ~l~c~rio~lly. A
30 voltamctric i~nmunoa~say can be ~a~r~ed by label~ng one immunor~ac~an~ w.~th oin ~lec~roactive ~ub~tance. Pace u~, Pat~nt ~o. 4~23~,144, i~ued No~ember 11, 1980, ia illu~txative o~ o~e ouch techni~aue. ~nother method involv~ ~arldwiching ~n anti~n-21ntibody lay~r be~ween ,, ... , ~ .;;., ..... ._. .. ....... . . .. . ,.. ~.... ....... .

' ~' .

~2--~2~
two conductive layer~ and me~ur1ng ~he ele~tri~al capa~itan~e of the result~ng l~minate. Gi~ever UOS.
Patent No~ 4,054,646, i~ued October 18, 1977, describes ~uch a metho~. .5 fur~cher ~ype of ~pa~tan~e-me~rlng 5 ~yst~m includes a pair of electrodes coated with a substrat~ and ~ rs~d in a medium containing a materi~l which 3p~cifically ~inds ~i~h the ~ub~tr~te, ~ ~less~ribed in Arwin ~. S . Pat~nt No. 4, 072, S76 . A further method ~ombines change ef~ect ~gr~al de~ect.i~n wi~h an enzyme 10 imrnunoa~ay techniqlle. ~uch a method i~ disclo~ed by ~ibbons U~S. P~tellt No. 4,2~7,~00, i~ued ~eptember l, 19~1. The foregoing electrlc!al rl~e~hods ha~, ho~eY~r, failed to provide medi~a~ pr~ctitioner~ d labora~ories with ~ ~imple, fast, ~ensltive, inexpen~iv~ and easy-to-15 u$e method of p~r$orming an immunodiagno6~ est~
one ac~pect of the present lnvent 1on involves~he u~e of antigerl or a~tibody-label~d ~olloi~al gold p~rt$ole~. In general, "colloidal ~old~' ref~ to a ~u~pen~ion of fine ~old particl~ ln water ~r a~ueous ~olution~ ~reparat~on o~ ~u~h pa~icles ie di~clos~ by DeMey, et al~ U.S. Paterlt No. 4,446,238, ~ued ~y 1, 1~84, and ~e~ey, et al. U.S. Patent ~o. ~,420,558, i~ued Decem~ex 13, lg~3. Such colloldal qold preparations have been prevlously used in immunodiagnostic tests wherein the results are determined optically by observing small amounts of light reflected as a result of the antigen-antibody reaction. The foreqoing patents to DeMey dis-close a bright field light method of the foregoing type.
Silver enhancement as been previously used as a means for staining gold partlcles. The present invention advanta-geously employs colloidal gold, optionally with silver enhancement, in a new immunodiagnostic method.

.... ~ .. _~ .. .... ... . ... .... ..

;~0~ 2~6~3 S~AR~ OP ~E ~ 023 The pr~ent inv~n~ion provi~es ~ method ~or detectin~ a binding reaction betw~en a p~ir o~ fir~t and second 3ub~t~n~es, parti~ulRrly bio~enic ~ub4tances, 5 s~hich speci~ically bind toge~:her. The me~hod o~ the lnvention involve~ hringing ~he ~ub~ar~es together ~o tha~ the blnding reactlon between thQn~ cAu~e~ full OE
p~rti~l completion ~106in~) of an es~entially open electrical cir~ui~. ~he resul~ g change in th~
10 elect~ical state o~ the c~ r~uit ir~dicates ~he binding reaction .
A~ording to a further a~pe~t of the inventlon, a dia~no~tlc elelnent for u~e in detec~in~ ~ ~inding reaction compri~e a p~i~ of ~pac~d-apart, ~lectrical 15 con~tor~, p~rti~ularly conduct~ve layers, di~posed on a ~ub~tantially n~n-ele~trically conduc~iv~ bahe. The ba~e may compri~e a support ~avin~ ~ ~ayer formed t~ereon which has a high ~inity ~or pro~ein ~inding and ha~
mod~rate to h~h ~6i~tance ln comp~ri on to the ~onductor6. ~e ~pace be~een the o~nductors defines a path or channel. one of ~ pair o~ eub~tan~s whic~ bind to each other ~s depo3ited on ~nd a~fixed to the ~inding l~yer of the non~conductive ba~e between ~he conduc~ors, ~uc~ a~ on the ~ottom w~ f a ~hannel. Mean~ forming 2s an electrical ~irc~it ls conn~ct~d ~o each of the conductors 90 ~hat the ch~nnel o~n~titute~ ~ break in the cl~uit.
As ~ ed here~n, the ~erm '1diagnosti~ element"
refer~ to the baBe~ conductor~, and l~yer o~ one o~ th~
binding ~ub~t~nces, without th~ me~n~ de~ining th~
el~ctricAl clr~uit, 9u~h a diag~oBtic ~lement and ~e~ns ~orming an ~lactri~al ~ircuit ~n r2~dily be us~d in ~on~un~ti~n wi~h any ~uitable m~ns $or ~ully or p~ially ~ridgiYI~ the bre~k in th~ ~ircuit du~ to the 35 binding reac~ion between the p21ir ~f ~ub~n~e6. One ~u~h Tnean~ involv~ ~dh~ring one o~ the subY,tance~ to the ~urfa~es of el~trl~lly condt~ re particl~s.

....... ;
~.............................................................................................................................................................................................
......

.

F DEBc~Ipq~Io~a 0~ ~WI~
Pre~erred exempl~ry embo~imen~ w~ll here~ter be described in ~on jun~tion w~ ~h the append~d drawin~, wherein like desi~na~ion~ denote like e:leme~ nd:
~ ures lA, lB, lC, 1~ and lE 2lnd Fl~ure~3 2A, C~ 2~ and 2E ar~ hc~ematic di~gram~ illu~tr~ting ~n immur~odia~nostic method accordislg ~o t~e inventiorl;
~gure 3 ~8 ~ ~he~tic dlagram illu~tratin~ a reaction dete~or ac~ording to one ~D~bodiment o~ the invqntiont E~igu~es 4A and 4~ z~e ~che~atic diagram~
~h~wing aggr~gate ~orma~ion aocordin~ to the me~hod of Figure~ lA--lE and 2A-2E, r~Bp~ctlvely;
Fi~ure 5 i8 a ~Lche~ratic diagram ~howin~ binding of a corsdu~tlve p~rtlcle to ~ non-conduc~iYe ~ase ~ccordirl~ 1;o one embodiment of the invention;
I`iqure ~ is ~ c~o~ ectional vi~ o~ a diA~no~t~ el~ment accord~ng to one embodiment of the invention;
~igure 7 1~ a ~o~ e~tlonal vlew of an alternAtive di~yno~tic ele~nent ~ceordin~ to the invention Figure ~ i~ a plan ~.7iew o~ a multiple diagno~tic elemer~t ~ccordlng to the invention:
Figu~e 9 i~ ~ plan view of an alternative nultiple dlagno~tic e~ement according tQ the invention;
~igure IO ~ ~ a ~chematic diagram o~ an un~oatea bound ~ggregate ~c~dlng to the inv~tion ~i~u~e 11 18 a ~h~rnatic di~ram of a co~lted b~und ag}lr~gate according to the invention;
~igule 1~ i~ a ~hematic di~gr~m o~ r~ tive ~huntin~ o~ ~urren~ ~acording to tP~ in~ ntion;
~igure 13 i~ a ~r~p~ whereir~ re~istallc~ values ater ~ilver ov~rcoating ~nd sil~r count~, both 813 obtained in Example ~ l ar~ plot~ ag~inst gold-antlbody ~on~tlgate dllution; and , 2010Z6~

Figure 14 i~ raph wher~in peroen~ o maximum refii~tAnc~ det~xmined ~ n Example 4 i~ plotted ag~ins~
~mount of ~ompeting ~ntigen.

~ETAI~ 8~ rIoN
~he method of the ~nvention is particularly u~e~ul ~or det~ting ~nt1~ens in th~ fluids or tissue~ of hurnans or anirnal~. Such antigen~ include drugs, toxin~, l~ormon~, allergens, tumor ma~lc~r6, f~ctors, en~yme~, ste~oid~, nucleotite~ and othe~ ~ub~t~n~e~ as li~ed in Elu~ng U.S. ~at~n~ Na. 4~3~71rO73~ isaued ~pril 27, 1982~, Any of the foregoing listed subs~ances can provoke the ; production of a reactive substance (antibody) which re-acts with and binds to the antigen~ Accordingly, the method of the present invention is useful for detection of a wide variety of substances which may be present in the living - body of a human or lower animal, for example, in drug over-dose trea~ment, where it is desired to quickly deter-mine which drug a patient has ta~en.

Fi~ure~ lA-lE ~chem~tic~lly illuRtrate 20 method ~or detecting ~n ~ntigen accordin~ ~o the pre~ent inv~ntion. ~e~erring ~o ~i~ure~ lA through lC, a p~tlent ~ample llA ~ lA~ ~uch ~B whole blood, blood ~erum or urine, aontaining a p~r~l~ular antigen 12A is mixed with a ~olloid~l ~old preparatlon 13A ~IG. 1~) con~aining ~ p~edetermlned ~ount of gold ~article~ l~A
having antibo~ies lSA fix~d t~ ~he outer ~r~aces tAereoE. Anti~odies 15A specifi~ally bind to antlgen 12A
(~IG. lC). In the r~ultiny mixtur~ ~6A, antig~ A
bind~ wlth available antibo~ie~ l~A, re~ul~ln~ in ~ree ~o~pl~xe~ 18A, comprislng bo~ antigen 12~ ~nd antibody 15A ~ound to pArticle6 14A. Since there ~re more antibodi~s 15A than anti~en~ 12A, sqme ~ntibodie~ 15A
r~maln ~r~ unbound to en antigen l~A.

-Z~ 6 Aa illu~a~ed ln ~igure~ 2~-2~ ~ the ~oregoin~
procedure i~ al~o carri~d out uslng a corltrol ~ample llB
(F~:G. 2) lacking the ~ntig~n 12A and a second colloldal gold preparation 13B ~FI~. 2B~ ~u~tantially identic~l in s compositlon to prep~ation 13A used wlth p~tient s~mple llA. The thus-~ormed ~econd mixture l~B (FI~. 2C) lacka the o~nplexe~ 18A ~hown in Figure 3.A, and ~orr~pondingly ha~ a greater num~er s: f part~ 3 h~ving unbound anti~en lS~ on the 6ur~a~e~ thereof . The f i~t mlxture 16A, corresponding to the patier~t s2lnple t~IG. lC), and the seoand mlxture 16B (~IG. 2C), corresponding to the ~ontrol, a~e ~hen re~dy for use wi~h a rea~tlon de~ector 20 ac~o~ding to the invention (~IGS. l~, 2D)o Referring now ~o ~igll~e 3, re~ction dete~tor 20 i ncludes ~ non-electrically conduc~ive ba6e ~2, ,a pair of ~hin, ~paced-apart ~leotrLoally ~onducti~e layer~ 2~, ~4 di~posed ~ide-by-~ide on ba~e 2~ with a ~hannel 32 form~d thereb~ween, and mean d~finlny an eleotri~al cir~uit, ~u~h as an ohmmeter ~fi functionally ~onne~ted ~o layer~
~o 23, ~4 as ~hown ~y means ~u~h a~ wire~ 2~ y~s ~3, ~4 act a~ a p~ir of po~itive and negative te~minal~ ~or the circuit.
~e~er~ g again ~o Figures 1 and ~, id~ntical flr~t ~nd se~or~d reactiorl dete~tors ~OA ~IG. lt~) and 20B
~ . 2D) ~re prepared irl advan~e or u~e wi~h mixture~
16~, 16B, re~pectively. Samples of ~ntigen in a c~r~e~
liqui~ ~eOg., wa~er or ~al~ne ~olution) ~e poured into 6helllow ch~nnel~ or groove~ 3~, 3Z9 def ~ r~ed bet~een layer6 23~/B and 24~,~ to cauae ~ntigen to ~ind to the 3u~ces of bot~om walls 33A,B of ch~rlnels 32A,B to ~orm antigen laye~ 30A, 3~B t ~IG~ . lP, 2D)~ The~e antig~n lay~r~ 30A, 30~ a~e made o~ the salne ~ype o~ ~ntigen a~
an~igen 12A ~o be d~te~ed.
Rei~rring now to ~igure 1~ t mlx~u~ 16 tFIG~ lG), cor~p~ndirl~ to patient ~ample llA t~IG-lA), 18 pou~ed into ~hannel 32A of ~ir~t det~ctor 20A to c~llse binding of ~nt~ge~ yer 30A snd antibody 15A. The foregolng pro~dure i~ o ~arri~d o~t u~in~ the control , 2~
_7_ mixture 16B ( lacking ~omplexes l~A3 and ~e~ond d~tector 20~ ~IG. 2E)o Condu~tiv~ p~rticles 14A h~ving free antibodie~ 15A thereon ~f~ectlvely become ~ound to bot~om wall 33A via antigen layer 30A due to the ar~tigen-5 antibody binding re~ction. Complexes 18A contalningantigen 12A from ~ample llA do not ~end to become bc~und So bottom wal~ 3~A.
After a ~u~ ta~le time to flllow the ~nti~en~
~ntibody intera~tion to take place, ~h~nnel 32A
10 i6 flushed wi~h ~ sultable liguid, e.g., ~t~r or ~aline ~ol~tior~, to wa~h ~way afly unbolund partlcles 14A, then dried by ~ny suita~le mean~, such as ~leating or 3110win~
the reaction d~te~tor to ~tand op~n to the air. The resi~t~n~e ~nea~urement can al~o be per~ormed wet, without 15 any drying st~p. Even when wet ~ ~he d~fferenee in mea~u~e~ re~lstan~e i 6uffi~ient to in~ Ate wh~th~r the bir~ding reaction has o~curred.
Non-~pe~ bi nding of particl~ to lay~rs 30A, 30B 006urs ~o ~o~e extent. Ordinary wa~hin~
~0 proc~dllre~ may not b~ ~u~ ent to remove 3uch non-~p~clfi~ally b~und pa~t~c~les from thE~ ~ntigen l~yerO
~oweve~, it ha~ been found that ul~ra~onic ~eatment of t~ie .~ ples at ~ low level can remove non-specif i~ally b~und particles without removing specifically bound 25 p~rti~le~ e. ~ parti~les bound due ~o antigen~antibody binding involving tne 61lb~tance being dete~ted ~or. This i~ pa~ticularly impGr~ant 83 a means o~ preventing a f~l~e po61tive re~ult. Su~h a f~l~e po itlve re~ult is a dec~2a6e in r~ tan~e due to non-spe~lfically bound ~0 p~rti~l~s, rather than specifically bound pfl~ti~les.
Referring now to Figur~f~ 4A and ~B, ~igure 4A
corr~6pond~ to the ~ame state a~ ure lE, ~nd F-~gure 4B
c4~re4ponds to the ~ame ~tate a~ F~gure 2E. F1gures 4A, 4~ illustr~te the diff~renc~s ln t~le extent of the 3S bind~n~ ~actlon n~ ~pp~rent in Flgures lE7 ~E~
Ohmln~te~s ~6A ~nd 26B regi~t~r the r~istance acro~
~harmel~ 3~A and 32~ ~or ea~h o~ the det~l;or~ 20A and 2013. ~or ~h~ ~ontrol ~E'IG. 4B), all p~rtial~ :L4B having . , . .. . . .. . . . _ , 7, . ; . . .. . .. , . . , . ~ . . . _ . . .... .

6~

~ree antabodie~ 15~ depc~ited thereon are av~ilable for bindlng wi th antlgen l~y~r 30~ ~ound ~o hatto~n wall 33B.
A6 a re~ult, ~ompl~xe6 35B are for~ned 2t bo~tom wall 33 ~nd ar~ an~hored there~o. A~ illuatra~ed in F$~ur~ 4~, S complexes 35B tend to ~lu~ter together in c~ontact with each ot~er to ~onn ag~re~te~ or ~hain~ 39'd which ef~ec'citrely br~d~e cbannel 3~B. Since particle~ 14B are ele~trl~ally conductiv~, a~greg~e~ 39B ~f~ectively provide ~n ele~trical connection between layers 23~ an~
10 24~, completing ~ elçct~ ir~uit defined b~ ohmmeter ~6~, wire6 ~3~, layers 23B, 2~B, ~nd aggreg~te~ ~9B.
This i~ re~le~ted by the re~ ance reading ~iven by o~meter 26~. ~ d~a~tic decrease ~n re~istance occurs a,s ~ result 4~ bridging of a~greg~te~ 39B, The ~eacti~n for the mixture 16~ correapc~nding to th~ patient ~ample proce~d~ in a ~imllar fa~llion, except that thi~ mixture l~A already ~ont~ln~ complexe~
l~A ~rme~ by reaction of an~lgen 12A with antibody 15A.
5inc~ the antibodi~s o~ the~e comple~e6 l~A ~re alre~dy 20 ~ound wlth at lea~t some antlgqn l~A, the~e complexes 18A
~o not tend to bind to the layer o~ antig~n 30A at bottom wall 33A. In mixture 16At the num~er of ree antibodies 15A aepo~lt~d on parti~les 14~ i6 les~ an in mixture 16B, s~n~e ~ome of the~e ~ntibodies 15~ were u~ed to form 25 complexes l~A. A~ ~hown its Flgure 4~, A~regate8 39A
~orm, bul; there a~e ~e~1er ~u~h a~g~g~tes, and corr~porldingly less b~id~ing ~ chantlel ~2A. ~ ~
re~ul~, the d~rea~e ln resi~t~rlce ~giBt~re~ by oh~ ter 26P~, if any, ~ ~ less than the d~cre~e in resistanc~
30 reyi~te~ed by ohtnmet~r 26B. This di~ference in reading~
ind~ate~ e pre~enc~ o~ antl~en 12A in the patieLlt ssmple llA. I~ patient ~mple 11~ doe~ not ~ontain any ~ntigen 12A, then the decr2as~ in reai~anae ~or reaction detect4~ alOA wo~ld be th~ ~me ~ th~ decre~se in 35 re~i~tan~ ~o~ d~t~ctor ~0~, ~ r~istance v~ll3e~ corresponding to ~ip~c~ic an~lgen l~v~l~ in. the ~unple are well known for al 0p~cific to~ e fore~oin~ proc~dure can be ~aEraed out .. . . . ...... ~ .. ~

~ ~ ~2~ 6 ~

withou~ the co~rol lllu~trated in Fi~ure~ , 4~.
~oweveri the u~ of a ~ontrol i~ pre~e~r~d becau~e the ~omparatlve r~a~tance re~dinga produc~d by th~ control afford more accurat~ ~esult~.
The pro~edure ~ho~n ln ~gures 1 through 4 is grea~ly ~i~pli~ied or purpo~es of illu~t~a~ion.
~onduct~v~ partlcles 14A,~ are l~rger ~n anti~en 12A
and antibodie~ lsA~s~ ~ nu~ber of 2nt$bodie~ 15A,~ are b~und to ~ s~ngl~ conducti~e pd~icle 14~ or l~s, and lo 6imilarly a number of ~ntigen~ 12A can bind with anti~odl~ 15A on the surf~ce of a sin~le particl~ 14A or 14~. ~igur~ 5 sc~ematically illustrates how a condu~tive pa~ticle 14B having ~ntibodie~ lSB on it~ surace becomes ~ound to bottom wall 33s via antigen layer 30B.
The method of ~igure~ 1-4 ~tilize~ a po~itive contr~l, thA~ is ~ . d~crea~e in resi~tance d~e to bridging o th~ channel b~ th~ ~onducti~e p~rticl~s OCCUr5 for the ~ontrol ~a~ple, not th~ unkno~n (p~tient~
~ample. ~he m~thod o~ the invention may al~o be ~arried ~0 out u~ing a ne~ative ~o~t~ol wherein t~e ~mple ~on~aining the ~nknown, for ex~mple, an ~n~l~o~y, ig reacted dir~tly with the g~ld parti~les, and then the go~d p~Yti~le~ a~e brought in contact with ~he reac~i~n urface co~ted with ~he corre~ponding anti~en. The procedure 1~ ~ub~anti~lly t~e ~ame a~ show~ in ~iyuxe ~B, ex~ept that the amou~t o~ the antibody i~ unkn~wn.
Many variation~ of ~he method of the inv~ntion, including both co~p~titi~e and non-co~p~itl~e pro~edure~, are poesible. ~he proc~s illu~trated in ~0 ~igs. lA-lE is a co~pe~ltive rea~ion ~hereln ~he anti~n is bound to th~ di~gno~tic el~en~, ~nd ~r~e antlg~n in the ~ample llA comp~t~ with the b~und antigen ~o~ ~it~s ~n the ~o~du~tiv~ly l~belled antibody. ~he p~in~ub~lon ~tep ~hown in ~g, 1~ may b~ o~itted, and 3S t~ ~ample 11~ ~n~ ~oll~ld~l gol~ 13A mAy b~ add~d dir~c~ly ~o ~n~lgen l~yer 30A bound t~ the ~ no~tic el~ment. ~lternativ~ly, t~e ~ntibody rnay in~t~ad be bound to the ~l~gnos~lc el~m~nt~ and con~u~ti~ly ... .. . ., , , _, _ lab~ d anti~en can ~ompet~ with free ~tigen ir~ th~
~ample for antibody binding ~ite~. In either ca~, the r~6i tance m~asu~ements are di~0ctly relat~d ~o tlle amount of ~r~e antig~n in the ~ample, in othar word~, the 5 m~a~ed ~e3ist~nce in~rea~e~ directly ~i~h inc~easing amount~ of ~ree antigen in the sample.
In a non-comp~iti~e varia~ion use~ul for antigen~ that ~an bind ~o more than one ~ntibody at ~he same time, ~ ~lr~t antibody is ~o~nd to the diagno~ti~
element in ~ predete~mined ~mount in exces~ of the amount req~ired to ~ind all avallable ~reP antigen in the ~amplQ~ The ~ample i~ added ~o the bo~nd ~ntibody and allowed to re~ct. A ~e~ond, conduc~lvely labell~d antibody 1~ ~dded, ei~her later or at the ~ame tl~e as lS the ~ple. ~he ~e~ond antibody al~o r~a~t~ with the antigen, re~ulting in a ~omplex compri~iny firat anti~ody-anti~en-second antibody-~ondu~tive pa~icle ~oun~ ~o the di~gnostlc ~l~men~ The ~wo bindln~ on the antlgen may be identi~ tru~turally dif~erentl or two-site ~o immunom~trio. ~he re~ultin~ resistanee mea~urements are nve~sely ~elated to the ~mbunts o~ free antigen in tl~e ~ample, such that the ~esist;qnc:e de~reases ~ the amount of ~ree ~nSigen ~ n~rea~e~ ~, Another non-comp~titive varl~tion i~ u~eful ~or 2S deter~nining eunounts oE apeci~ic ~ntibody ~ a aample, pa~ti~ul~ly an ~ntibody titer for a dl~ea~e or allergy.
The antigen i~ bol~nd to the diagno~t 1 c element in ~ufficl~nt e~ce~s to birld to ~he ~nt~ody is~ proportion to i~s concent~tion. A ~ample containing ~r~e antlbody 30 i~ add~d to the bound antigen and i~llo~d ~o r~act, A
secondary an~ibo~y c~rrying the ~onductive particl0s 1 a~d~d, ~ithe~ l~ter or at ~h~ ~ame tin~ a~ the ~ample.
~he ~eeondary ~n~lbody rea~t~ with the ~ir~t An~ibody, i,~., t~t6 lt a~ an ~ntigen, ~orming a compl~x 3~ oompr.l~ing ~n~igen-fir3t ~ntibody~econd antibody-conductlv~ part~le. The ~ec~nda~y an~i~ody could be, ~or ~x~ple, anti-immunoglobulln G ~ E. Th~
r~sulting re~i~tan~e m~urement~ ~r~ lnve~sely rela~ed .. . . . _ . . . , _ 66~

to the amount of fre~ ~fir~t) an~iboày in t~e sample. A
~econd~y antibody ~an al60 be u~ed in embodiments whe~ein ~e~istance ohange i8 dire~tly r~lated to mea~ured antigen ln the ~atnple in order to in~rease ~en~itivity, Ba~es 22A, 22~ di60u~sed above mus~ have a highly biorea~tive 3urface. "Bioreactive" as dis~us~ed her~in refers ~o the ability of th~ sllr~ace to bind biogenic ~ub~tances ~u~h a6 protei.ns or nucleotide~.
~ioreactivity values meA~ured for variou$ organic ~nd ino~ganic ~ub~tances vary wldely e~t~en among chemically sim11ar ~ub~t~nces. ~owever, ~ertain pla~tics ~nd ~netal oxlde~ and nitri~e~ are generally bioreactive9 In a serie~ of enzyme immunoassay experiment~ e E~eample 3 ) the ab60rption o~ light at 490 nanomete~s ~as used standard for determlninq bindin~ by a r~bblt IgG-~oat anti-rabbit IgG ~omplex to ea~h ~urface ~ted. Nylon 6~ had the highe~t level of bioreaotivity mes~ured. For purpo~e~ of the pr~sent ~n~er~tion, s~lbstan~e~ having a biore~ctivity at least 70~ ~h~t of Nylon 66 for proteins are ~onsidered h~hly ~iorea~ti~e. Co~pound~ having ~bout ~0-70~ uch biore~otivity 8~ Nyl~n 66 are mildly bio~ea~tive, compound~ h2ving 10-50~ &re somewhat ~iorea~tive, ~nd composition~ h~vin~ a biorea~tivity of 2bout 10~ or le~ are bioinert.
It ha~ been ~u~d th~S the following substances are highly bioreactive wlt~ prot~in~: Nylon 66, polypropylene, mylar, ~hromium oxide, phenolic pla~tic, pOly~tyrene, and vinyl. Chromium oxide i~ unu~ually high ~n p~te1n bioreaotivity for a metal oxide. Moderately ~0 bioreactive ~u4~tance~ for p~otein6 include ~hromium, ~i~anium oxynit~ide, nickel oxide, ~antalu~ nitri~e and carb~n. Poorly blo~ea¢tlve ~u~tance~ ~or proteins in~lude titaniu~ oxid~, boron nitride, ~nd ~llicon oxide.
Biolner~ 9ub~tanc~ ~4~ prot0in~ inolud~ ~ilicon nltxi~e, barlum tit~nium ox.id~, indium tin oxide, ~lu~inum oxide ~nd gl~

.

.

6~

~ igur~ 6 and 7 illustrate two diagnostic element~ 10~, 10~ th~t can be used in ~he me~hod of the in~rention. In Figure 7, dia~no~tis el~ment ~OA a~cording to the invention includes a ~upport 40 ~u~h as a glas~
5 plate, e~g. ~ mi~roscope slide, ~oated with a th1n layer 4SA of an ~l~ctrically re~istive, bior~active subst~nae ~uch as chromium oxide, ~ p~ir of conductive lay~r~, gUCh 26 ~hromium m~tal lay~r~ 51, 5~, are ~upe~posed on the re~i~t?ve, biorea~ive layer 4~A. A ~hannel 4~
~ep~rate~ layer~ 51, 52. A lay~2 of an anti~n 53 6pan5 ch~nnel 42 l ~nd may a,l90 C:OV~ yers 51~ 5~ . L~yers 45A~ 51, 52 are preferahly formed by ~put~ring on a gla~ plate. ~hann~l 42 i~ then formed ~y photollthogr~phy u~ing An etchant tha~ selec~i~ely ~taçks the condu~tive layer but doe~ not attack layer 45A. In thl~ di;~gno~tic element, tlle width of channel 42 rela~iYe to the ~ize of the condu~tiYe particle~ to be u~ed is important ~ec~use th~ b1ndln~ rQ~ction Ls prim~ily indi~ted by ~omplete brldging o~ channel 42 by ~g~re~aat~g of th~ condu~tlv~ p~rticles~ ~he resistive layer ~5A 1B highly rksl~tivet i.~ ~nore than about 106 ohmS ~m, ~o that virtual}y ~o ~hange in resi~tance is ~au~e~ ~y partial bri~ging a~ ~hown in Pigure 4A.
F~gure 6 illus~ra~e~ an altern~tive diagnostic element wherein ~onductive layers 51, 52 ~re for~ned dlreatly on ~upport 40, with channel 42 ~ormed therebetw~en. Resi~tive layer ~5B i8 ~uperposed over suppor~ 40 ~n~ layers 51~ S2, and arltigen l~yer 53 is then formed ~hereon. In ~his embodi~aent layer. 4S}3 l~
only mode~at~ly re~istive, e.g. lO3-106 ohm~-cm~ 50 ~ha~
a ~u~Qtanti~l ~h~nge in re~is~nc~ Wlll oo~ur due to r~ tive ~hunt~ng of current through aggreg~:e~ bourld to l~yer 53, ev~n wh~n rao complete bridgin~ occur~.
E'lgure l~ 1llu~trete~ tor through whlch re~istiv~ ~hun~ing oc~U~. ln s~kln~ th~ p~th 71 o~
l~a~at r~ai~tanc~, the curr~nt will be ~hunted through bo-~nd ~gr~g24~5 72 w~ch o~er a lo~e~ siot~nce thAn the ~u~port 40~ The overall resi~anc~ drop ~cro~s ~;

.

.

2~

channel 42 will dep~nd on how ~sany ag~regate~ are bound, ~nd can provide ~ ~uanti~ative indica~ion o~ the a~ount of binding being me~ured. In t}~e ~e3i~0r shown, the calculated resi~tance drops f~om 7D,000 to 40,030 ohm~
5 followln~ the rea~tion.
~ he wid~h o~ channel 32 or ~2 may vary, parti~ula~ly in relation to khe ~imple numer ~ cal average di~me~er of the conductive p~rticle~ fc~rming a ~hain to bridge the channel. The f~llowing t~ble ~tes preferred 10 ran~e~ Eor dimension3 f~r gap-bridging embodin~ent ~cording to the pre6ent in~ention;

TA~31.~ 1 ~verage Ratio of P~rti~le Ch~nnel Channel Width lS D1ame~r Width to Partiele 0.01~500 D.l 20,000 S;l to 40:1 Q.01-10 0.1-1~0 10:1 ~o 30:1 ~,01 1 1-~5 lS~ 2~:1 2~ A 20 :1 ra~lo of ahannel width to avera~e par~icle diameter i~ ~ypi~al, e~g., the ~hannel ha~ a width o~ lo micron~, and th~ erage di~meter o the ~onduot ive p~ti~les i~ 0.5 mi~ron6. q'he foregoing ~nge~ are also ~eul in re~istlve ahunting embodiment~ t mu~h 2s larger ~hannel widths may be ~mployed, ~u~h 4~ Up to 1 mm, 1 cm, or ~e~ter, d~pending on tlle de~ir~d applio~tlon.
A~ an a.lternsti~7~? to a ohannel, diagnostic ~lements acco~dl~l~ to kh~ invention, ~6p~c~11y r~si~tive ~ ~huntin~ ~mbodlm~nts, Inay utillze a re~i~tlve~
blor~a~ti~re p~th oth~ than ~ ch~nnel. Such a path m~y conlp~i8er 1~0~ ~x~mple, ~ ~ur~ed line which 6p~n~ the cond~or.4 but doe~ n~t represen~ the ~horte~t dl~t~ce b~twe~n them. Similarly, the oonduc~or~ n~d not be in 4~6~

the form of l~y~r~ Sm~ll wir~ ~uperpo~ed Ol~ the ba~e can, for ~xample, be elnploy~d a~ the conductor~.
Protein~ h~ve an ~fflni~y for msterials ~u~h a~
poly~tyrene, c~r~miu~ oxid~ and the llke, and t~nd to beco!ne bound t~ereto under ~uitable ~onditions. Protein~
~an ~l~o read~ly become bound to the ~urfaces of f i ne metal p3rSi~l~s, ~uch ~s gold particles, using the procedu~ e descri~d below. In em~odi~r ent~ of the invention which in~olve antigen-an~ibody binding~ it is preferred to bind the ~ntigen to the bot~om wall of the ~hannel and bind ~he ~ntibodies to the condL~ctive parti~l~s. }lowever, the re~erse arr~ngement (antigen-particle~ r antibody-chAnnel ) can al~o be employed .
Conduntive layers 51, 52 In~y have any de~ired di~en~ion~ whi~h prot~e functional. I~o red~ce the ~i2e of the di~gno~tic ~lementr theae layerOE ~re generally ~s thin ~ po~6i~1e, and pre~er~bly have a thicknass no grea~er than about 5 mic~on6 " p~ee~ably no greater than abo~t O . 5 mioron~, particularly a ~hickness in the r~nge of 0,ool-0. 005 mi~on~ . Conventional ~putter depo~ition can b~ ~e~dily u3ed tb for~ the eondu~tive layers in any desi red ~h~pe.
Layerfi 51, 52 (or 23, 24) may be ~ormed of any ~uitable c~ndu~ti~e m~teri~l, pa~ticularly an el~tric~lly conducti~e metal 3u~h a~ gold, silver, platlnum, ~opper, ~romiu~ or al~minum. Particles l~A, 14B are prefera~ly ~ade from ~ ~onductive met~l such as g~l~, silve~, or pl~tinum, and may alqo be m~de of car40n pl~tel~t~ or pla6tic p~rticl~ having a conductlve metal co~tin~, e~peci~lly g~ld-coated p~ly3tyrene ~pheres.
Suc~ coated ~pher~s ar~ llght~r than oompar~ble solid ~t~l sphe~e~ and are thue bett~r able to maint~in bon~in~ to t~e su~f~c~.
'rhe ext~n~ oE bindin~ o~ an an~ibody to metal 3s p~rti~le~ i8 infl~nced by vario~s f~cto~. A ~erie~ of en2yme immunoa say ~xperlme~t~ demonstrated that af~inity-pur~fl~d ~ntibody te~d~ ~o bind ~o gold particle~ to a mu~h g~e~ter ext~nt than impu~, wh~le ~erum antibody, and ~hat the p~ o~ the ~y6~e~ a~o had a ~tron~ effect. ~ ~lightly a~idi~ pH (e,g. ~ 7) prod~ed ~eve~al-~old greater binding ~han a ba~ic p~ of 9-10 for l~r~e ~greater tha~ ~.5 ~m dia~eter) particle~.
Polyethylene glycol treatment of the gold parti~les may al~o be us~d i~ needed to st~bil~ze larg~ gold partiçles;
see ~orisberger et al., J. Rl~to~hem ~ , 25:~g~-3~5 (1977)~
The antibody concentration on the gold particle~ should be hlgh enough to allow the an~igen-antibody ~eaction to occur on the eurface~ o~ th~
gold partlcle~ ~o ~n extent suf~icient for de~ction by ; the me~hod o~ th~ invention. On the other hand, if the an~ibody ~oncentration i~ too ~reat, the ~ntibody layer 1~ may ~ave an in~ulating e~ect whl~h will re~ult in a ~al~e negative r~sult, i.e., will block the drop .in resi~an~e ~hat would nor~ally o~ur, unle3s ~n additional ~tep of over~oati~q the ~g~re~ates with conductive m~al i6 u~ed, a~ described hereafter.
Ba~e 22 de~crlbed ~bovQ iB ~de of a biorea~tiv~ plastic, preferably polypxbpylene, mylar, phenolic pla~tic, vinyl, methyl cellulo~e, ~ylon or poly~tyrene. Tbe polari~y of protein mole~ule~ causes such molecule~ to bind to 6uch a plastic ba~e to form a sub~tantially ~omplete, homogenou6 coating o~ antigen.
~lass by itsel~ is not generally employed a~ the ~a~e (support) ~ln~e anti~ens have a poor afinity for a gla3s ~urf~ce and it h~s p~ved dl~icult to ~d~ere a layer o~
an~igen ~o ~ gla~ slide. ~owever, accordi~g to a ~urth~r aspect of the invention, it has b~en found that 91~ slide c~n be ~urfac~ tre~ted 80 that antigen Afinity ~or the ooating on the gl2~ slid~ ~ecom~s a gr~at or gra~ter than anti~en ~f~ni~y ~or ~ c~nventlonal poly~y~ne ~lld~. As noted above, thl~ ~ur~ace ~r~Atment compris~ ~oating t~a gla~ ~upport 40 with a thin lAyer 45A or 4S~ o~ a ma~erial havin~ ~ ~oderate to high el~c~rl~al re i6t~n~e a~ ~ompared to the cond~ctive layer~O The re~is~snce o~ this l~y~r i8 i~ the ran~e . ' 6~

103-108 ohm~-cm~ prefe~ably lOq-la7 ohm6~cm, ~ compared ~o a re~istan~e of le~ th~n lO0 ohms-cm ~or the conductors. Mod~rately resistive mat~r.ial~ for purpo~e6 of the inveI~tion ar~ tho~e having me~ured re~ nce~ in 5 the r~nge of 103 up to 1~6 ohmY-cm ~h~n ~posited on a gla~ pport, wherea~ highly reaisl:ive materials have compar~ble mea~ured ~e~i~tances of 106 or mo~e.
aiorea~tiv~ sub~tan~e~ sui~abl~ for forming layer 4SA or 45~ include ~arbon, hydrophilic organi~
10 polymer3, and lnorganic met~l oxide~3 and rlitride~. Such ~nor~anic met~l co~opounds include oxlâe~, nitrides and oxynitride~ o$ boron, alumlnum, silicon~ cadmium, copper, nickel, ~ob~lt, iron, mangane~e, ~nd metal~ of Group~
IIA, ~IIB through VIB. ~pecially prefe~red inorg~nic 15 m~t~l compound~ incltlde chromium oxide lCrO3) r tlt~n~um oxyn rid~ ~TiOXNy), tantalum ni~ride, c~rmet materi~ls such as chromium/silicon oxide ~cr~Sio) or gol~ on oxide, and other iTIorganic ma~erials Qurrently used in r~ tor6. ~ydrophili~ o~gan~c polymer3 include well-20 known pla~tic~ such as mylar, polystyrene and nylons,~u~h a~ Nylon 5l;.
~ f chromium oxide 18 us~d as lay2r 45A, i~ iY, u~ually deposited in the form ~f CrO2. To m~ke it more biore~c~ive, the layer nnay be ag~d in ~he pr~enc~ of 2S oxygen and water vapor ~o convert it to CrO3, which is hydrophillc. Thi~ ~ging can be accelerated by placing ~he co~ed ba~ or fini~ed di~gnostic element in a humidi~y ch~mber . Carbon ~n any convenient foxsn, e . 9 .
graphite, is ~ theor~tically i~eal material for l~y~r ~5 30 in v~w of itg biocompatability, although it~ thin film ~e~i~t~nce rn~y be h~rd ~o control ~ithin n~rrow ~an~e~.
Cermet r~ to~ ate~ial~ h~ve a high re~l~tan~, ar~ ~t~ble, and hav~ lo~ negative tenlp~rature coe~icient~, ~uah that the dia~no6tic el~ment will not 35 be ex~es~iv~ly temperatur~ ~ensi~:l v~ . Wh~n hea~e~ in ~i~, cermet ~ oxidlze and incr~a~e ln re ietance, and are u~ually provided wi~h a protective ~ver~oat:in~ to .. . . _ . .. . ._ . j .. . . .... . . . .

!

prevent oxidation ~nd~or hydr~tlon. In the pre~ent inventlon, however, layer ~S doe~ not re~uire ~uch a p~ote~tive ~oating.
~ i~ure ~ illustrates ~ fur~her e~bodiment of the diagn~ti~ ~lement of the invQntiOn wherein ~
~ultiplicity of reaction si~s are dispo~ed on a single non-conductive ba~e 22. Each ~eac~ion site c~mpri~e~ a pair of condu~tor~, 6u~h a~ layer~ 23, ~, ha~ing a layer o~ a bio~nic sub8tance ~hic}l undergoe~ ~pec~ic bindiny dl~po6ed in the ohannel or p~h therebetween. Th~
biogenic sub6tance, 6~ch a~ an a~tigen, may ke the ~am~
or d~f~erent ~r each rea~ti~n site, ~nductive mean~ ~8 for this embodiment compri es a ~eri~ of individual el~c~xi~al ~ondu~tors 61 Whi~ e~ch connect to ~ common co~duc~or 6~, which in tu~n conne~ts to a te~inal plate 63 mou~t~d on the edge of ba~e 22. Pairs of l~yers ~3, 24 are arrayed in ~OW6 and column6 on ba~e 22.
In the embodiment o~ ~ig~e ~, ~ow~ of pl~tes 63 a~e di~posed on adjaa~nt ~id~ of re~angular ba~
22. ~o preve~t overla~ping hetween a fir6t set b~ of condu~to~s C2 ~onnec~ed to lay~rs 2~ and a ~eoond ~et 67 of condu~tors ~ ~o~nect~d to l~y~rs 2~, all but one of conductors 62 of second set 67 are lo~ated on the rever~e side of base 22. The~e conduc~or~ 62 ara illustrated by ~5 brok~n l~es in F19ure a. ~uter c~ndu~tor 62A doe~ not cro~ ~ny conductor~ 62 of ftrst ~et 66 and thus doe~ not need to be on t~e oth~r side of b~e ~2, alt~ou~h it ean be so located if de~i~ed. Indlvidual oonducto~ 61 connect~d to common con~u~tor~ G2 of se~ond ~et 67 o~ the re~r~e side of base 22 in~lude portionæ 6~ ~hich ex~end.
through the thi~kne~ o~ ba~e 22 ~d ~onne~t with su~h c~nduotors 62, ~hown by brok~n line~ in ~ig. ~.
Oh~m~ter 26 can be conn~t~d to vario~s ~ombin~tion6 o~ terminal pl~te~ 63 to ~eas~rç resl~tan~
fo~ ~aoh p~ir o~ layars 23, ~4~ T~ make su¢h a ~e~ur~ent, ohmmeter 26 i8 ~onn~cte~ to two plates 63 on dl~r~nt edges o b~ 2. Connecting an ohmmoter to plate~ ~3C, 63P would test ~he indi~ted pai~ of. lay~rs 2~6~

23C, 24D. Th~ 8 smbodim~nt allo~ a single diagnostic ele~ent a~cording to the in~7ention to t~t a ~ingle patient sample for a num~er of different ~ub~tances, ~ucl a3 antigen~, since e~ch p8ir of layer~ 23, 24 can have a 5 dif~erent s~b~tance bound therebetweexl, or h~ve no 6ub~tance bound therebetw~n ~o ~g ~o provide a control.
In t~e alternativer ~he ~ame -~u~st3nce may be ~ested ~or a number of time3 to provid~ a more certain r~ult.
In an ~lternative embodiment ~h~wn in Figure 9, n ~err~in~ls 63 are disposed alon~ only one ed~e o~ b~s~ ~2.
One t~rminal 63E i8 conn~c~ed ~o eaoh layer 2~ by ~ingle, multib~n~h~d ~onductor Ç2~. A seri~s u~
ter~ninal~ 63P are ea~h ~onne ted to one of layers ~ by ~epar~te ~ondtlctor~ 6~. Condu~tors 62~,~ are ~rranged lS to not croe~ ~ach other, 80 ~hat all of conductors 62E,~
~re dl~p~ed orl the front ~a~e o~ the diagno~tic e~emen~
~ ~holNrs. For thi~ pu~po~e terminal 63E is located ~t one end of the row of spaced-apart ~erminalR ~3~,~. Thi~
embodiment ~voids th~ n~ed to ~rovide wires, ~on~uctive 20 line~, or ~he like on bo~h s~idE!Y of the plate, and allows the ~erminal~ tb ~e di6posed on a common ed¢~
Re~i~tive shunting ~c~ording ~o the 3nethod of the inven~lon ~ay be greatly enhan~ed by ove~coatin~ the ~greg~tes wi th a l~yer of a conducti~e ~ubst~nce, ~5 parti~ularly a me~al ~uch a~ sil~e~f gold, or pl~tinum.
~he applied ~?a~ing S~ sticlc ~ele~tively to the aggre~at~ but not to the remain~er of t~e the path betwe~sl the conducto~s. If gold par~lcle~ ara u~ed a~ the particles ~o which one ~f the binding ~ub~tar~c~s are 30 bound, ~;lver enhancement may be used to orm a c~ndll~tlve ~ er coa~ing over the ~g~regat~.
Condu~tive met~l o~tercoatln~ i~ e~pecially u~e~ul ~or re~istiv~3 ~hunting embodiments of ~tle inventi~n fo~ two reason~ Fl~t, i~ can eliminate 35 pr~bl~ tlt~ iun~tion re3l~tan~e e~ect~. Referring to ~igllre 10, Tesi~tiv~ ~hunting 1~ minimi2ed by the relatlvely ~nall contact ~u~ e of the a~greg~ 7fi with the underlyin~ layer 53. ~n addl~ion, the bio~eni~

66~
--lg-coa~n~ 77 on th~ gold p~rticles 7~ c~rl for%l an inl3~1atin9 b~rrier ~uch ~ha~ th~ junction xesistan~e Rj i~ hlgh enough to pr~t~ent re~isti~e shuntirlg. By contra~t 9 th~ ~onductive met~l coating 7g ~hown in ~i~ure 5 11 crea~es a much lower re~i~t~nce ~ and allow~
resi3tive ~hun~ing to ocour. Thi~ embodiment o~ the lnvention i especially pl~eferred becau~ it improve3 reliab~ y, as demon~trated by kh~ re~ul~s of ~xample 2 belo~ .
Ssoonà, m~tal over~o~tin~ ~an eli~ninate the nee~ to use par~icle~ which are both bioreactive and conduo~iv~. Sinc~ the metal o~er~oatin~ can ~ond~c~ ~he cllrrent~ ~he pa~ti~le~ can be ma~e o~ a non-conductive stlb~ance, ~.g., rlon-conducti~e beads may be u~ed 50 lon~
15 a~ the metal overcoating ~tick~ ~ele~tively thereto.
Thi~ ~dvantage also pert~ to gap ~ridging embvdiment~
a~ well.
Th~ following exampl~s d~ribe a ~ap bri~ging embodiment ~Exampl~ 1), A -re~istive 8hunting 20 elnbodl~ent ~Example 2 ) and ~n ex~ ple o~ a procedure ~or de~er~nining bior~otiYil:y a~cording to the inventiorl ~xample 3 ) .

~AM~L~3 1 A. Preparation of ~
The followi~g p~ocedure wa~ u~ed ~o makle a su~pen~ivn o~ gold parti~l~s. ~la~sware inluding ~ flA6k and stirring ~ar were ~lean~d by ~oniGs~ion in b~th sonicator, ~irst in ~tha~ol, then acetone, ~hen pe~roleum etber, ~or about 5 mlnLI~es ~ach. ~h~ glas~ware 30 wa~ th~n l~own dry with a Fr~on r~ig~rant, then rin5~d ~wice with doubl~ gl~s~-d~Rtilled water. About 1 ml r ) of ~ (weigl~t/vol~ame) giltered ~olu~lon of ~old ~hlo~ide in w~ r and 99 ml of filtered, doubl~ ~la~ dl~ d water wer~ ~dded to the clealled 3S fl;ask. ~he r~sultin~ Jnixture ~as th~n h~a~d and stirr~d wlth ~he cle~n~d stir~ing b~r until ~ol~ g. Al: th~t time 300 ~1 o~ a 1~ ~v filtor~d ~olution of l:ri~odiu~

.. . . _ _. ... ,_.. , , .. _ . ,, ., , ,,_, ;_,, _ , .

~lt26~
--~o--citr~te in w~ter wa~ added. Bolling wa~ corlt~nued with q~irring, and a~ditional 1~ tri~adium cl'crate ~olution wa~ ~dded in amollnts o~ 100 ~ 50 and 50 ~1 at int~rval~ of 5, 12 and 15 minutes, r~pe~tively, from the time the 5 mix~ure began boillng. After 30 mtnutes o~ bolling the ~la~k wa~ removed ~rom ~e~t and sllow~d to cool. The average particle ~iz~ o~ the re~ul~ing ~ollo~dal gold particle~ wa~ lnO n~n a~ deter~ ed u~ing a ~ran~mis6ion elect~on micro~cope, ~he fla~k conta~rling ~he colloldal 10 gold preparation w~ ~ealed and ~tored at 4 B. ~ ~
About 9 ml of the colloldal ~old prepar~ion nade in part A above, 100 ~1 o~ ~.2 M potas~ium earbonate and ~0 ~Ll of a p~otQin~ an a~finlty-puFifled goat anti-15 rabbit immunoglobulin (~gG) antibody, were added to a 15mi coni~al cent~ifuge ~;ube. ~he ing~ed~ents w~re mixed . c~verni~ht on ~ tu~ rota~or. 1 rnl n~ a filtered agu~ous polyethylene glycol ~olu~ion ~2 mg P~/ml) wa6 then ad~d as a ~tabilizing ag~nt. ~ ration in thi~ ~nd ~o ~ub~e~uen~ pro~dures wa~ ~arxied out uslny a 0.2 mi~ron filter diæk fitted into a ~yrin~e. The re~ulting sold-prot~ln ~on~ug~tes were concentra~ed by centrifugation ~t 11,000 rpm ~or 10 minute~. The ~upern~tant wa~
di~oarded, 2nd the re~ulting pellet o~ gold parti~le~ wa~
pipqtted into ~ 1.5 ml centri~uge tu~e. Th~ pa~ticle~
were ~h~ wa~hed by ~n~ri~ugation four tlme~ with a pho~phate bu~r~d ~aline ~olution (0.01 M ~odium pho~phate di~ic, 0.15 ~ 30diu~ chlor~de, 0.1 mg/ml polyethyle~e ~ly~olr p~ 7.4) ~nt~ining 0.2% ~odiu~n a2ide a~ ~ pre~ervat~ve to re~ove unbound protein. ~he ~onjug~ted gold particle~ were th~n re~u~pended ln 1,4 ml o~ phoaphflt~ bu~red ~line ~lution c~n~aining 0.2%
~odlum aæide.

~30Z66~ ~
-21- !

C. ~
glas6 311de ~8 coa~ed ~ith A thin layer of ~hromium oxide tCrOx, where~n x=1-3~ by radiofrequency ~ r~) magnetron ~put~er deposition u~ing ~ ~aterial~
~es~arch ~orporation Model 822 Sputt~rsphere. A chromium pl~te target was used as the cathode. The glas3 slide wa~ placed on a copper pla~e anode dispos~d beneath the cathode. Deposition wa6 carri~d out unde~ the following condit~on~: ~y~te~ ~ase pre6sure ~ x 10 7 Torr, tot~l pres~ur~ 8 x 10-3 Tor~, ~orward power l,O~OW~ target voltage ~35V, re~lected power 0 w~tts, cathode to anode di tance 2~ inche~, ~nd a sputtering time o~ 30 minutes.
~he y~tem wa~ back~illed with oxygen~ Th~ de4ired chromium oxide layer h~in~ a thi~kn~s3 of about 0,1 ~m w~ thereby formed by deposition in the oxygen pla~ma. A
lay~r o chromium was ~hen d~po~ited over the chromium oxide layer by repeating the ~oregoing procedu~e in an inert argon pla~ma, re~ulting ln a urfa~e l~y~r of c~romium having a thickness of 1 ~.

D. _o~gll~hg9~bY
A line ha~ing a width of 5 ~ wa6 then ~ormed ln ~he ~ur~oe chromium layer of the double-coated ~lide of p~t C ~y photolithography. In yellow room light, layer of photoresi~t pla~tic wa~ irst ~or~d over the chr~mium l~yer a~ follows. æhe twice-coated ~lide was placed in th~ chu~k of ~ H~adwAy Model EC101 ~pinner unde~ v~c~umr About 3 ml o~ Shipley lllS p~otoresist was applied over the ~hrom~u~ layer and allowed to ~p.r~ad for 10 se~nds. ~e ~ e was ~hen ~pun at 2~00 rpm .Eor 30 ~0 ~econd~ to Eorm a unifor~ thi~k photore~ifit c~ating.
Several ~u~h photore~i6t-~oatçd ~lides wer~ then baked in a ho~ plat~ o~en on a rack ~or about 20 minut~ at 80C, then ~llowed to oool ~or 5 ~lnut~s.
A ~u69 M~55 photolithograph~c mask ~ligner wa~
3S calibra~d and prep~red or u~e. A ma~k defining a 5 line wa~ cleaned ~horoughly wlth dissolv~d ~oap and w~ter, then d~ied with a nitrogen ~tream that lleft no ~.
.

realdue. ~he ma~k wa th~n pl~ced in a Tn~k holder under vacuum, and ~he ma~k h~lder wa~ ~hen pla~d in poQi~ion ~n the aligner. The triple-coated ~llde Wi~15 placed beneath th~ 5 ,um }ine of the m~k. The maBk holder w~
5 then locked in plac~, and the ~ligner was used to expose the photore~i~t layer to W light ~or 18 se~ond~. The exposed slid~ wa6 ~hen remov~a f rom the align~r .
~ he s}ide~ were then inunersed in a re~h 4 :1 developer solution ~ 4 par t~ w~ter ~o 1 part Shipley 303A
10 develop~r ) Xor 2 mlnut~s w~th no agitation, and then p~omptly r~n~ed ~ith distill~d water. ~ach &lide ~as ~hen iTnmers~d in the etc~ant, a solution h~ated t:o 4~C
con~i ting of gO.8 g A1~13, 27.0 9 ZnCl~, 6 ml ~3PO4 ~nd 80 ml distilled wa~er ~ ~or about 10 ~econds~ During thi~
15 tisne ~he etchant re~o~ed undevelop~d pho~oresi~t materlal and ~hromium Inetal along ~e 5 Inicron line which wa~
pr~viously expo~ed to l~V light. ~owever, ~he etc:hant did not a~a~k the chro~nium oxid~ layer. ~fter etching, the slide wa~ pro~dptly r~rlsed in dis~llled wa~er. ~he 20 photore~i~t material wa~ therl removed from th~ ~urfa~e of ~h~ chromiu~n layer~ by immersing ~he slide~ )r about S
minutes irl Shipley lll~A r~mover. The~ft@r the slide wa~ rins~d thoroughly ~n t~p wa~er, then ~in~ed ultra~onically in di~tilled w~ter ~or 10 mlnutes. The 25 ~lide was then re-immer~ed in the remover, aTId thle rinsing ~tep9 were ~epe~ted to en~ure that the photore~i~t m~teri~l wa~ co~pletely removed~

E, Me~uremen~' ndin~eaction rrhe re~i~tanCe o~ ~he din~nostic ~lement5 30 prep~ed in part D w~s ~nea~ured using ~n ohmmet~r. Each el~ment wa~ then pla~ed in a te~t ~u~e ~nd rin~ed with a coating buf ~er ~ 0 . 015 M sodium ~arbona~e, 0 . 0~5 M ~odium bica~l~on~t~ 0.003 M sodium azide, pH 9.~, Pairs of diagnostic ele~nents were prepa~ad by ~dding either rabbit 3~ immuno~lc~bulill (IgG) or bovine serum ~lbumin tB8A) to eac~ te~ tu~e. ~he ~l~m~n~ were incuba~ed by allowing th~ ~ubea to st~nd overnlght ~t room ~emp~!3r~tur~e. ThQ

. .. . . . _ .. ... .... .. _ .. . . . . . .... .

~3 66~

protein-coat~d elements w~e then wa~h~d three time6 wi~h a PBS-~riton buf~r (pho~pha~e bu~fered 821irle ~olution a.B described above conta1ning 0.1 vol. ~ Triton X-lO0 88 .
a wetting agent ) to realove exce~s unbound protein . The 5 coated sub6trates were then placed on a piece o~ parafilm in ~ petri dish. The colloidal gold~protein corljugate wa~ then ~pplied to each elem~nt in an amoullt Ruf f ici~n~
to cover the upper su~face of ~ach element, ~nd the element~ were allowed to incubate ~tand3 for 20 minute~
10 at ~oom temperature, ~h element wa~ then washed by ~piration once ~n PBS-Triton buffer and once 1n distilled water, and then ~llowed to air dry for l hour.
~he re~i~tan~e of each ~lement was the~ nlea~ured using the same ohmsn~ter a~ init~ally u~ed to mea~ure the 15 resist~nce of e~ch element.
All element~ ~e~ted had a ~tarting resi~tance ~reater th~n ~ mlllion ohrn~. Positive re~ult~ were obtained for ~amplen at line w1dths of 3, 5, 6 and 7 . 5 ~lm. F~r these ~mples, the m~as~lred res1~tance at the 2Q ~econd m~a~uremen~, ~ollowing ~he ~ ti~n, dropped to v~lu~ ranging ~rom 85 to 700 ohms. A few IgG sample~
re~ulted in weakly po~itive result~ ranging f rom 80, 000 to 800, 000 ohm~ . Negative r~ul~cs were obt~ined ~or the 3SA sample~ 1 the resi~t~n~e ~ter the ~eaction remained 25 higher th~n 2 m~ llion ohm~ xoept that t~o weakl~y po~it~ve rc ults for ~8A #a~pl~ were obtained at ~he 5 m lin~ ~i8e.
5imilar result6 were obtained when the fQ~e~oing procedure ~as rep~ated at d~ ~ferent 30 temper~tures or in~u~ati~n times. ~sually, a po~itiv~
result was ab~ained ~ith a rea~tion tlme of ao minute~.
The~e variations o~ the proc~d~e indi~te thAt the re~ction time c~n be aho~tened ~ub~tantially.

,.., . -r -- :

.

6~

~PI.E 2 Protein labelled ~olloidal gold W1~8 prepared a~
5 des~xli~Pd in ~xample 1, par~ ~ an~ ~. A diagno~3tic ele~2nt according to the invention wa~ ther~ prepflred by first coating a gla~ mlcro~cop~ ~lid~ with a ~hin l~yer o~ o~r~mium by r~ m~gnetron ~putter depo~ition using the pro~edure of Ea~ample 1, part C, Qac~pt that no 10 in~erveni~ layer of c~aromlum ox~de wa6 ~ormed. The ~h~o~nium-~oated ~lide wa~ then ~ubjected to pho~olithography a~ d~cri~ed in ~x~mple 1, p~rt ~, except tn~t the line formed had a width o~ 1 ~nillimeter.
A ~hin l~yer of carbon wa~ ~h~n deposited o~er 15 the enti~e upp~r ~urf~oe of the ~lid~, a8 ~hown in ~igure 6, by sputter d~po~ition a~cor~ing to Exampl~ 1, pa~t C, ~x~ept a~ ~ollows: ~he ba~kfill ~as was ~r~on, and the tar~et ~dthod~ wa~ ~ a gr~phite dl~k 8 in~he~ in di~m~t~r and O ~ 25 inch ~hick . ~he ~raphi~e di~k wa3 20 epoxyed to a water-~ooled copp~r ba~king plat:e.
D~po~itlon was oa~ri~d out at 1~5 V ~i~h ~ ~orward pow~r of 200 W. The d~61~ed c~rbon lay~r havln~ a thickn~s~ of ~bout O . 25 ,um wa~ thereby ~ormed by deposition in the argon pla~ma.

B. ~
The reY,i~tanc~ o~ the ~agno~tic elements p~ep~red in part ~ wa6 mea~ur~ u~ g ar~ ohmm~er. Each el~ment WB9 t~en pla~ed ln a t~8t tube and rin~ed with a ooatirlg b~f~er ~p~ 918~ a~ d~cribed ~bo~). Pairs of 30 po~itive ~nd n~ti~e diagno~tic elom~nt~ were prepared )~y addillg ~ith~r r~bbit ilnmunoglobulin ~ ~gt;~ or b~vine ~erum ~lbumin ~ A) to eaoh te~ tube. q~he ~lemQnts were ubat~d by allo~ing th~ tub~q to ~nd overnight q~
room ~cemperat~re. The prot~aln-caate~ el~ments ~ere then 35 ~a~hed ~hre~ ~im~ w~th ~ P~-Tritorl bu~2r ~ho~pha~e buf1ared salinY ~olu~ion ao d~s~ribe~ abo~ta oon1taining 0,.1 vc~ Trlton X-l~O a~ ttin~ agent) to re~oYe ., . . .... .. . , ,__ .. .... ... ... . ...

Dz6 --~5-~xce~ unbound pro~ein. The ~oa~ed sub~ta~es were ~hen pla~ed c~n a pie~e of par~llm in ~ pe'cri di~h. ~he colloidal gold-protein conjugate wa~ ~hen ~?plied to ea~h ~lement in an amount ~u~fic1ent to aover ~he upper 5 ~urface ~f each ele~nent, ~nd the elements were allowed to incu~te (st~nd~ for 2, l or ~ hour at r40m temp~rature.
E:a~h element was then wa~hed onc~ in PBS-q?ri~on bu~fer and on~ in distill~d water I and then ~llowed to air dry ~or 1 hour. Irhe re~i~tan~e of each element wa~ ~hen lQ mea~u~:ed u~ing ~h~ 6ame ohsl4neter a6 initially u~ed to measure ~he re61stan~ oi~ eac~ elemen~.

C. Silver Enhancement . . _ Th~ el~rnent~ prepared in part B above were rewashed wit:h di8tilled water to ~emove ~hlsrlde ion~
15 ~hqt might I~act ~ith the sil~er enhan~em~nt r~age~nts. A
2 M ~odium citra'ce buffer ~olutionl a 0 . 5 M hy~roquinone ~olution and a 0 0 03 M ~ er l~ct~te ~olution wer~
prepared ~ n a d~rkened room, The thre~ reagen~ re tllen mixed ~ogetl~er to pro~ide 25 nll of overcoating reagent. The e1ement~ were ~omp1e~te1y i~nmer~ed in thi~
reagent ~or ~3 minut~s, then lnus er~ed in a 1 % a~e~ic ~id 301ution for 2 minut~6, ~nd ~hen imlnersed in a f1xat1~e ~o1ution ~or ~or 2 n1inut~a. ~he fixative used wa~ Rodak Rapid ~ix, ~ont~ining anmlonium th1Osu1f~te, ~5 a~tic acid, ~odium m~tabisu1~ite, s~dium tet~aborate, and alunlinum su1fate. Th~ e1emen~s were r1nsed ln di6tilled water ~r 10-15 minutes and a11Owed to ai~ dry.
The re~1~tan~e ~f e~ch element was then mea~u~ed ~ing the sa~e ohmmete~ as u~ed in ~amp1~ ~, p~ t B .
3û ~he reai~tance re~u1t~ re stim~rized in ~ble 2~ In part B, 8~n~p1es 6A, ~B were ir~ubated for 1 hour, 9arnp1es 7~, 7~ were incub~ted ~or ~ hour, ~nd the ~naining ~amp1~3 w~ incubated ~or ~ hou~.

~t~rti~g R After ~ After R Gold ~ r 96 ~ !Ohm~
-lA(EISA)298,000394,000 30~,000 ~D.00 lB( IgG)357, oon444, ooo 379 _~9 ~ ~
2A( E18A)1, 060, 0001, 2~0, OûO950 ~ -10 . O
~3(IgG)1,110,0001,~7û,000 6,B00 9g.1 3A~B~A)4,gOO,OQ06,210,~004,050,0D0 -17.3 lû 3B~ IgG)5, 4~0, 0006, 560, 0001, 410, 000-74 . 2 4A~ A)39,~00 71,500 43,~00 ~ 9.3 Ig(~)3s,aoo 5~,30D 1~2 ~ 5 SA( BSA)3, 600, 00~3, 600, 0002, 600, 000-27 . ~
513(IGG)1,100,0001~100~000 6D3 _~9.9 hA~E~5iA)48,600~15,200 71,Q00 ~46.1 6EI~IG~;)72,5001~0,3nO ~Z0 -9~.7 7~(B~A,)2~,~00 $0,300 41,400 ~4E)e8 7EI~ IGG )42, ~0069, 400 6~0 -~8 . 4 ~n increase in ~ ta~o~ of abou~ 10-loO~ normally 20 o~curs ~hen the c~rbon ~11mB of ~x~mple ~ are expo~ed to w~ter~ ~t a ch~nn~l width c~f 1 nun, no nomplete bridging occurs whl~h migh~ ~u9e a decr~a~se in re5i~tance.
~owev~r, ~e~istive ~llunting embodilnent~ accor~ing ~o the invention can 6ucceesfully detect a binding rea~tion even 25 ~l~hout me~al over~ating i~ a narr~w ohannel width lor ~horSer pat~) ia u~ed, ~nd other re~ction condit~on~ a~e adjll~ted ~ccordingly, D. ~ltrasoulld P~ocedure A pellr ~f ~S~ and IgG diagn~tic elements were 30 prep~red acc4rding to ~e p~oc:~d~ e o~ E~ mple 2, p~rt B, usir ~ A 2 hour inoub~tion per1Od. A~ter ~ hour~ both ~ur~ces were w~l~hed ~nd dri/~a~ A high level O~ gold p~rticle bindl~ w~ ob~rv~d mi~o~oopic~lly ~or l:he IgG
~Anlplet and a mode~at~ to low amount o~ bir~ding was 35 ob~orved or th~ B8A ~mpl~ ~oth ~an~ we~e th~n i~mner~ed in dl~till~ed ~w~ter ~Jla ~ub~e~ted to 1 In;Lnute of ~2~6 treat~ent with ~ Vi~30ni~ 50 Virti~ cell d~ pter at its lowe~t po~er ~et~$ng. The ~ampl~ ~ere ~hen removed and reexamined, No dif~erence in the amount o~ gold binding was observed ~or the IgG ~mple, wherea~ mo~t of the previou~ly ob~erved gold particles on the ~ s~mple were removed ~y the treatment. In another experiment in which a ~i~ilar ultra~onic tr~a~m~n~ was used, a f~lse positive ~ample (~ BSA sample for whl~h ~ drop in re~i~tance wa~
noted) o~ the ~ype de~oribed in Example 1, p~rt ~, w~
converted to a negative ~mple.

~XAMPLE 3 The sen~tivlty of ~he method of ~he invention wa~ evalu~ted by determining a llmiting am~unt of antibody reguired for an a~ay. DiagnoBtic elem~n~ were prepar~d by cutting conductive polyimide plastic (K~pton cont~ining blended carbon) lnto 1 ~m by 0.3 c~ squ~eq.
The piece~ wer~ rin~ed onc~ in ~oa~ing bufer tde~rlbed above) and imme~ed overnight at room t~pera~ure in t~t tube aontaining elther 1~ ~SA in coat~ng buf~er or 5 20 ~gfml r~4bit I9G ~the antigen)~ The resultin~ ~ample elements were t~en washed three times with P~S-~riton sol~tlon (de~cribed a~ove) and plac~d on par~fil~ in petri dishe6. A ~olution of goat a~ti-rabbit I~G
antibody bound to ~old particles, prepared sccord:ing to t~ proc2dure o~ Example 1, Part~ A and ~, wa6 ser~ally diluted, and two drop~ ~t ea~h dilution were appli~d to o~ ~SA-aoate~ sa~ple and one Ig~ ~o~ted ~ample. After 2 hour~ of incubation at room temperature, t~e 32mple~ were fir~t wa~h~d with PBS-~rlton ~olutionl and then w~th di~tilled w~ter. Th~ ~mple~ w~re t~en allow~d to dry, ~nd the re~i3~no~ o~ ea~h ~ple waa ~a~ured. Silver overcoating wa~ then ~arrie~ ou~ ~or each s~mple u9i~g the pro~Ur~ o~ Example 2, part C. ~he ~si~tance of e~a~ ~ampl~ wa~ th~n meAsu~ed, ~d kh~ nu~ber of silver count~ at ~ach dilution wa~ det~rm.lned by x-ray analysi~.

, . . . . .... ~ . , ., .. , .... . .. ... _ . . .

26~

The ~ult~ were a~ :Eollow~, ~herqin re~i~tanc~
value~ ~) a~e giverl in me~aohm~ e~pt wh~re noted:

~ 3 S~ple/ R ~f~erR ~t~r S~lver ~ ~P~ d ~ Count~
lA ~ BSP~3 Non~1. 55 2 . 0~ 1. 46 lR t IgG) None1. 4~ 1. 72 11~ ohms2861 ~A (B~A~ ls21~S2 2.11 1~28 2B ~ I~G ~ 1. 47 - 21~7 1~ 3A (BSA) 1:4~.52 1,94 1.67 ~B t IgG) 1; 41. 53 1. 57 0 . ~1 1723 4A ~B~A3 ls~1.41 1~60 1.43 4s ~ 1 1.,57 0.54 1174 SA ~}~SA) 1~161~ 40 1~, 5 5~ 9 1.~7 0~2 74 ~A ~B8A) 1O321.~ 5 1.4~ -~B (IyG~ 1;321.47 1.82 1.0~ 6~9 Resi~tance ~alues aft~r 6~1ve~ ove~coating and ~ er count~ obtained in thi~ ~x~mpl~ ar~ plt~tt~ a~n~t gold-20 antibody ~onju~ate dllution in Fi~ure 13, whereln X'~~pre~nt sil~er count~ ~nd clrcle~ repre~nt res.i~tance in megao~ he lim~ting antibody concentration was ~ound to occur a~ a dllution o~ about 1:~ The re~lst~nce ~nd ~ilver oount valueq 13sth ~aried ~5 ~ub3tantially linea~ly wi~h c~ncentr~tlon, ~nd the change in ~e~is~an~e was oun~ to vary inver~ely w~h the n~lmber o:E ~ilvor count~, d~mon~tr~ting that the el~tent of re~istive shuntlng wa~ direct~y r~lated t~ the nu~nber o~
silver ~v~rco~ted a,ygr~gate~ bound ~o th~ ~u~ce~

!' ' ' .._. ... " ,,_ _ _ . :

~LE 4 ~ l~e ~en~itivity of the method of the inverltion was further evaluated by gene~ating ~ dose re~pon~e cu~ve. ~i~go~tic ~lements were prepared by ~utting 5 conductive ~apton pl~6tic a de~c~lbed in Exdlnple 3 into Z03 ~m by 0.3 ~m pieceQ. Titanium lnetal ~ont~ct pads . (0.~ ~y 0.3 ~m) ~e~e di~posed ~t opposite end~ of e~ch strip 1. 3 ~m apart to form resistors . The resi~tan~e of each resi~tor was then mea~ured. The resistorg w~re 10 r~n~ed once wlth co~ting buf~er, incubated in r~b~it Ig~, and washed Shree time~ u~ing ~he procedure of Ex~mple 3.
~ilutions of rabbit IgG in ~oating ~uffer conl:airling rabbit IgG in t~e ~mount~ given in T~le 4 below were prepared and prein~albatPd with gold-goat anti-rabbit IgG
15 c!oniu~ste pr~pared ~c~rdLn~ to Example 3 at ~ 1: 8 dilution ~or 11. 5 minuteY D The pre~ncubated con jug~te wa~ tllen app:Lied ~o the sample resi~tor~ disposed on parafil;m in petri dishe~ ~nd allowed to in~u~ate for 2 hour~ at ~oom tempera~ure. Th~ 6ample~ wer~ thcn wa~hed ~ onc~ wi~ PB~-Triton solutio~ nd ~nce with di~tllled water, and allow~d to dry. ~he re~i~tAn~e of e~ch ~ nple w~ tllen measuxed b~ connectin~ an ohmmeter to e~h of the cont~c~ pad~. The s~p1e~ were then ~i1ver over~o~ted a~ de~ribed in Exampl~ 2, part C, and the 25 re~ ance of the 6amp1es w~s aS~ain mea~ured.
The re~u1ts were a~ fo110ws, w~erein ~he re61stance value4 ~) are exp~esse~ in ki1001~ms and the amoun~ ~f rabbit IgG ~antig~n) i~ exprefl~d in nano~ram6:

. ' ~ .

;

i6 R~b.Ig~ er ~ ~ter %
~unt ~Q Gol~ ~ilv~r lA1000 311 3~9 335 ~ 4 lBloO0 318 355 340 - ~ . 2 2AS00 391 410 395 - 3 . 7 ~!~500 347 ~75 351 - 6, 4 3A100 347 3l33 ~4q~10 . 2 3B100 350 410 3~20, 5 4A50 371 ~06 371- 8. 6 4B50 376 416 385- 7 . S
SA10 3a8 434 368-15. 2 5B10 370 444 39~-10.1 6A 5 437 515 443~14 . 0 ~ 5 39~ 49~ 39~-~0 . g 7~ 98 17~. 3 7~ 1 392 480 g9-79 . 4 Figure 14 ~raphi~ally depi~ts a plot of percent o~
maxlmum r~si~tance ~100% minus percent change f rom I~bl~
20 5) vergu~3 the amount o~ çompeting ~ntigen, uAlng the averh~ o~ the ~wo re~ults ~, B obt~ined ~or ea~h amc~unt of an~igen plotte~ ~he re~ults indi~ate th~t the Inetho~
of thi6 example wa~ ~ensi~ive enough to ae~e~t a~ little as 1 n~r~og~m of an~l~en in ~ ~mple.

-- . . ....... . .. . .

Claims (10)

1. In a method of detecting a substance in a test sample, which substance is one of a pair of substances that undergo a specific binding reaction with each other, including the steps of reacting said test sample with a a reagent comprising the other of said pair of substances and determining the extent of reaction therebetween, the improvement which comprises:
contacting particles having a first one of said pair of substances bound to the surfaces thereof with a layer of the second of said pair of substances which substantially spans a path between a pair of spaced-apart electrical conductors superposed on a substantially non-electrically conductive base, each of said conductors being connected to means forming an electrical circuit which includes said conductors and said path therein, the binding reaction between said first and second substances causing said particles to be bound to said bottom wall to form aggregates;
removing unbound particles;
coating exposed outer surfaces of said aggregates with an electrically conductive substance;
and measuring a change in the electrical current flow through said circuit caused by formation of said coated aggreagates on said path, said electrical change indicating the level of said substance to be detected in said test sample.

2. The method of claim 1, wherein said step of measuring a change in current flow further comprises measuring a change in the electrical resistance across said electrical circuit.

3. The method of claim 1, wherein said particles are gold particles, and said of coating said aggregates further comprises treating said aggregates with a silver compound-containing coating reagent, treating the silver coated aggregates with an acid solution, fixing the silver coating, and rinsing the coated aggregates.

4. The method of claim 1, wherein said step of removing unbound particles further comprises ultrasonically treating non-specifically bound particles and washing said path.

5. A method of detecting a substance in a test sample, which substance is one of a pair of substances that undergo a specific binding reaction with each other, comprising the steps of:
(1) mixing, in a liquid medium, electrically conductive particles, said particles having a first one of said pair of substances bound to the surfaces thereof, with said test sample containing an unknown quantity of the second of said pair of substances, under conditions effective to cause binding of said second substance in said test sample to said first substance on the surfaces of said particles;
(2) disposing said particles on a substantially continuous layer of said second substance bound to an essentially non-conductive base to form aggregates of said particles bound to said layer of said second substance, said layer of said second substance substantially spanning the width of a channel between a pair of side-by-side, spaced apart conductive layers superposed on said non-conductive base, each of said conductive layers being connected to means defining an electrical circuit including said conductive layers and said channel;

(3) washing away excess unbound electrically conductive particles from said layer of said second substance;
(4) coating exposed outer surfaces of said aggregates with an electrically conductive substance which adheres selectively thereto; and (5) measuring a change in current flow through said circuit caused by the presence of said aggregates in said channel.

6. A method of detecting a substance in a test sample, which substance is one of a pair of substances that undergo a specific binding reaction with each other, comprising the steps of:
(1) mixing said test sample containing an unknown quantity of a first one of said pair of substances with electrically conductive particles in a liquid medium, under conditions effective to cause binding of said first substance in said test sample to the surfaces of said particles;
(2) disposing said particles on a substantially continuous layer of the second of said substances bound to an essentially non-conductive base to form aggregates of said particles bound to said layer of said second substance, said layer of said second substance substantially spanning the width of a channel between a pair of side-by-side, spaced apart conductors superposed on said non-conductive base, each of said conductors being connected to means defining an electrical circuit including said conductive layers and said channel;
(3) washing away excess unbound electrically conductive particles from said layer of said second substance;
(4) coating exposed outer surfaces of said aggregates with an electrically conductive substance which adheres selectively thereto; and (5) measuring a change in current flow through said circuit caused by the presence of said aggregates in said channel.

7. A diagnostic element for use in detecting a reaction between a pair of substances which specifically bind together, comprising:
a electrically resistive support comprising bioreactive particles blended in a plastic material;
a pair of spaced-apart electrical conductors superposed on said support; and a layer of one of said pairs of substances disposed on said support along a path which spans said conductors.

8. The diagnostic element of claim 7, wherein said particles are made of carbon.

9. The diagnostic element of claim 7, wherein said plastic is polyimide.

10. The diagnostic element of claim 7, wherein said layer of said substance is selected from the group consisting of microorganisms or portions thereof, hormones, allergens, tumor markers, enzymes, steroids, and nucleotides.