CA1223639A - Assay for nucleic acids - Google Patents
Assay for nucleic acidsInfo
- Publication number
- CA1223639A CA1223639A CA000470321A CA470321A CA1223639A CA 1223639 A CA1223639 A CA 1223639A CA 000470321 A CA000470321 A CA 000470321A CA 470321 A CA470321 A CA 470321A CA 1223639 A CA1223639 A CA 1223639A
- Authority
- CA
- Canada
- Prior art keywords
- assay
- mediator
- enzyme
- ligand
- antiligand
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/004—Enzyme electrodes mediator-assisted
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/817—Enzyme or microbe electrode
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/806—Electrical property or magnetic property
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/904—Oxidation - reduction indicators
Abstract
A B S T R A T
ASSAY FOR NUCLEI ACIDS
The present invention discloses an assay for nucleic acid which comprises the steps of;
(a) providing a probe material comprising;
(i) a sequence of nucleic acids complementary to a given target sequence and, (ii) a first ligand chemically linked thereto and capable of a specific binding reaction with an antiligand;
(b) contacting the said probe material with an assay system comprising;
(i) a suitable mediator, enzyme, substrate system capable of transferring charge to an electrode surface when the enzyme is catalytically active, and;
(ii)a second ligand chemically linked to one of said mediator, enzyme or substrate, wherein the second ligand is capable of a competitive binding reaction with the antiligand, and;
(iii) the said antiligand, whereby the said first ligand completes with the said second ligand specific in a specific binding reaction with the antiligand, and;
(c) contacting the above system with a solution suspected of containing the said target sequence whereby the binding of any of the said target sequence present to the probe affects the availability of the first ligand and therefore alters the rate of charge transfer to the electrode.
Refer to Figure 1
ASSAY FOR NUCLEI ACIDS
The present invention discloses an assay for nucleic acid which comprises the steps of;
(a) providing a probe material comprising;
(i) a sequence of nucleic acids complementary to a given target sequence and, (ii) a first ligand chemically linked thereto and capable of a specific binding reaction with an antiligand;
(b) contacting the said probe material with an assay system comprising;
(i) a suitable mediator, enzyme, substrate system capable of transferring charge to an electrode surface when the enzyme is catalytically active, and;
(ii)a second ligand chemically linked to one of said mediator, enzyme or substrate, wherein the second ligand is capable of a competitive binding reaction with the antiligand, and;
(iii) the said antiligand, whereby the said first ligand completes with the said second ligand specific in a specific binding reaction with the antiligand, and;
(c) contacting the above system with a solution suspected of containing the said target sequence whereby the binding of any of the said target sequence present to the probe affects the availability of the first ligand and therefore alters the rate of charge transfer to the electrode.
Refer to Figure 1
Description
? ~ 39 The present :inuention .is concerrled w.ith assays for llUC].eiC aci.(ls, the said assays extc!ncl-ing to both ch~tect.ion of the presence of nucleic acids and to the parti.cular i.derlti.fication of specific nuclf.!ic acicd rllolecllles .
7he.r(:! i.s a particu].ar need in may areas of biocherrlical reseach alld collllrlerce to be able to assay for the pr-esence of nucl.eic acids i.n a salnple and further to assay for the (?xist~nce of particular sequences within t ~ l i. CI ~ c ~ c l C i. Cl s .
Ih~! c!xp].oi.t.i~Liorl of t:hc! hybri.ti i sa ti.-Jn bc!Lwc!c!n t.wo single stranded DN~ or RN~ rr~ lecull?s wh:ich haue a corrlpl.errlc!rltilry sc!cluence has be~n on~! approach to this probleln. ~olecules for use in such assays which are capahle of binding to a DN~ or RN~ sequence which is def.ined l:o a particlJlar le~el of holnology are known as DN~-probes.
I<nown ~N~ (RNn) probe Lechiques share a simllari-ty in thi~t the DN~ (RNn) polyrner is not readily detectable by its i.nher~nt biocherl)ical acti~i.ty. It is therc!~ore ~q necessary to mark the polymer with some signal-producing chernical or biochernical species, such rnethods as are presently known include the following prior ~rt:--PRIOR ~ Rl h~ldln-Biotin_ R~actlon; this technology reli.es ~n the aFf~inity of the egg-whltc! glycoprotein a~idin for bi.o-ti.n. Biotin (Uitamin ~1) can be coval~!ntly linked to the rucleotide residuc!s which cornprise the rnonolneric subunits oF the r~Nh polymer. The modified subunits can still undergo the classical binding reaction between complementary strands of double~stranded DNh, and thus can be incorporated into syn-thetic DNh probes. ~t pr~!s~!nt, to clf~t-!ct the pr~!s~!nce of SUC~I probes which ha~e forrned short double-stranded regions af-ter exposure to comp].errlentary sample DNh, the unbo~rid probe must Firs-t be separated frorn the sarnple I~N~/bound probe complex. rhis is norrnally done by perforrning the binding reaction in conditions under which the salnple DNh is immobi].ized on a substrate and washing, although cen-trifugation rnay perform the sarne function. The bound probe is dete-cted by -the addition of a~idin to which either a fluorescent- rnarker~labeled antibody or an enzyme has been attached.
one problem with the above method is that small . ,;
J,3~
oligonucleotide probes (20 nucleotides) contain only a small number of biotinylated sites, limiting the amount of avidin which can be bound. Attempts have been made9 with some success, to add long "tails" of up to several thousand bases to the probe DNA, in which case only the tail need be labeled. The method can detect up to a resolution of 10 139 of DNA, or about 105 copies of a single gene. Although originally the marker on the avidin was horse radish peroxidase (a rather short-lived enzyme) the method has now been extended toinclude alkaline phosphatase. Unfortunately the method is generally difficult to establish for a new diagnostic scheme as either the biotin-linked probe DNA is difficult to prepare to the labeled tail interfers with sensitivity.
USE OF MEDIATORS:
Genetics Internationals' Canadian Patent Application 453584 of 4th May 1984 discloses a method in which at least one of a mediator and an enzyme are chemically linked to a nucleic acid probe sequence whereby speciflc binding of the probe sequence to the tarqet sequence in a single-strand nucleic acid material to be availability of the chemically linked species as detected by a sensor electrode in presence of` the enzyme substrate, whereby thepresence of the target sequence 3~
can b~ dett~ctecl ~s di.sclosed in the spc!cification the nuclei.c acid sequence can be RNh e.g messergt~r RN~ but is usually DN~.
sui.tablt! rnethod for the practice of thi.s techniqut-! has been giuen as follows;
(a) pro~iding a singlc! strand nucleic acid material to be in~estigated for a gi~en target sequence;
(b)s~:!].~cting a probc! rrlatc!ri~l with cl st!quc!ncc of nuclt!ic ac.ids cornplelnentary to the target seque~nce;
(c) choosi.rlg a proced~lr(! frorn ~mong;
.
(i) chelnically linking the probe with an enzyln~ and acldi.ng the enzyrrl~ linked probe to a solution containing both a substrate For the enzyrnt~ and a rnediator (ii) chemica]ly linkillg the probc! with a mediator and adding the mediator-linked probe to a solution containing both a substrat~ and an enzymt.! For the saicl substrate;
(iii) chemically linking thc! probe with a mccli.ator/
, 3~
enzyn1rl combination and adding the so-modified probe to a solution containing a substrate for the enzyrne (cl) contacting the solution containing the chemically linkecl probe sequence with a sensor electrocle whereby chargc! is transfc!rrr!d hy tl~r! mr!di.ator to thr! r-Jlr!ctrodr~
Froln the enzyrne-catalysed substrate reac-tion and (e) contacti.ng the soluti.on with the sirigle strandr!d mat~rial, whereby alteration in the a)nount of chargr.! transfr.!rred i5 an lndication of a specific binding reaction between thr! probr! and targr!t affr!c-t.ing thr.! a~ail~bi].ity of enzyrne rnediator or cornbination.
Ihe probr-! matr.!rial can br.! a naturally occuring [)~n fraglnent or a synthetically produced ~rlaterial.
~lterati.ons i.n the sequence of s-tr!ps can bre readily en~isaged. ~lso the sensor electrode itself can include the mediator or the r!nzyrne although genr.!ra].ly it is pre-ferred for the probe sequence anrd the tar~qc!t sequence botll to bc! prr!sc!r~t i.n sol~ltion.
rhe rnediator can be linl<ecl indlrectly to the probe sr-!quencr.! by a ].i.nker group and a nlaterial reacti~e to 3~
thc! ~.inker groups can b~! pr~!s~!r~t on th~! ~!].c!ctrod~! In this case the whole cornplex is present on the electrode;
when a target sequence is present, and binds to the probe sequence, an altera-tion in elec-trode current is produ c ed .
ME[)Ih~OR~ _)ISCLOJE[~, Th~! U S~! of rut~l-!ni.~lTrl corrlpl-!xes on a graphitc! ~!lc!c-trc)dc is cdisclosed in l<o~al and hnson [~naly-tical Chernistry, Uol 50 No 2, Fc~!b 1978 pg 223~. In which the follooing ruthenl.uln cornp~lexes are cliscussed;
Ru(NH )3-~2-~
Ru(NH3)5py Ru(NH3)5L
Wher~e L i5 pyridine, 4 aminorrlethylpyridine (hMP), or L
is N-(4 pico1inic)~-benzamide (PBh) -Ru(NH3)50~2 ' Ru(NH3)5~13~'2~
E RORS OF METh OLISM
':.,' 3~
One airn of the DN~ probe technology ancl the enzyrne dc!tection/assay tc!chnology so fal~ dc!~c!].opc!d has bc!c!n to detect inbuilt errors of rnetabolisrn which lead to a uai^lc!ty of ~I y~!nC!ti.C Cli,sC!clS ~!S and i.rlhc!rittlb]c!
disorders. ~rnong such clisorders arc-l: farnilial Goiter (i.odc)tyrosirlt! dc!ha1f~g~!nclsc! clc!fc!ctivc!) Maplc! syrup ul^inc disease (alpha--l<eto clecarboxylase deFective) Xanthinuria (Xanthine oxidase defective) and Me-th~3lnoglobinelrlia (Methelnoglobin reductase defestive).
f~l11 list of 3500 conditions duc! to d~!fc!ctivF.! gc!nc!s can be founcl in McKusick's "Menclelian Inheritance in Man".
Onc! partic~l].ar disc!asc! of i.ntc!rc!st is sicklc!-cc!ll amelnia in which one of the norlnal Globin qene coclons corl^esponcling -to the f:ifth sixth and seventh arrlino ac:ids of a particular chain are not as norlnal CC~-C~G-G~G but read Gcr--G-rG-c~G/ thereby rep1acing valine for glutarnic acid at position six. 5yn-thetic o].i.gonuclc!oti.d~!s ha~c! bc!c!n rrladc! which can act as probc~s For the sequence ~aria-tion in prena-tal diagnosis of the disc!asc! and succc!ssfully distinguish bc!tlAJc!c!n t~le wild type and the aberant gene. Assay of the colnponents of the binding rni.xture was perForrlled by a radiot,lc tiue tracer rnethod ~ sirnilar approach has been tal<en with the point mutation reportecl to cause antitrypsin ~, ;3~
deficiency.
~s can be seen frorn the aboue, howeuf!r, the methods of cletailed rnetabolic assay presen-tly auailabl~ haue certain disacluantagf!s i.n that they requirfe somf! or a].l of the followlrlq; short lifetirne reagf!nts (e1ther l~adi.oactiuf.!, ai~ s~!rlsiti.uf! or~ l.i.yht-sf.!llsitiuf!)~ lligh1y trained staff or (for autoradiography, ESR rnf!asurelnents or lc)w-~lf.!ue]. 1i.gllt clf!tf~c-ti.on) f!xpf~nsi.uf-! f-!qui.pmf!nt.
Known rne.-thods of assay -for binding reactions other than antigf~n/ant.i.bo(:ly and DN~/cornplf:!rrlf~ntary DN~ are genf!rally sirllilar tc) the aboue rne-tllods and s~lffer -Frorn simlldr prob].f!ms, ~ccorclirlg to onf-~ aspfec-t c,:f the prf!sf!nt inufenti.on thferf!
ls prouldfed an assay for n~lcll?ic acid which cornprises tll f ! C; t f ! p S C~ f;
(a) proui.ding a probe matf?ria]. corrlpri.si.ng;
(i) a sequence o-f nucleic acids cornplernentary to a giuen targfet sequence and, (ii) a fi.rst li.gand chc-!trliccll].y li.nked therf~to and capable of a specific blndinq reaction with an arti].igand;
(b) cont~cting the said probe matf!rial with an assay ;3~
systenn cornprising;
(i) a suitable mediator enzymc! suhstrate SySt.eTn capablfs of transferring charge to an tslectrode surface when t.he enzymt.! is catalyti.caly acti~e and;
(ii)a sfscond ligand chern.ically linked to one o,'- said TTIediator f.!nzyTTIf! or substratf! whc!rf!in thf! st~!cond liyand is capab1e o-f a competitiue binding reaction witll -the antil]gand and;
(ii.i) the said antiligand whereby the saicl first ligand cornpett-.!s with thf! sai.d second l.igar)cl in a specific binding reaction with -the antili.gancl and;
(c) contacti.ng thf..! abo~e systenn wi.th a soluti.on suspected of containing -the sa:icd target sf!quence whereby the binding of any of the saicl target sequt!nct.! prt.!st.!nt to the probe a-ffects the a~ailiabil~Lity of the First ligand and therefort.! alters thf! rate of charge transfer to the electrode.
By elTIploying a mt-!thod as described abo~e i-t is possi.ble to perforlrl the assay with an alnpl.iflcation step thereby i.ncreasing the resoluti.on of the assay.
Furtherlnore the rnethod of the present in~ention dcles not require the short li~c-!d radioactive assay components which are ernployed in othfsr types of assay.
;3t?3~
Enzyrne~substrate pairs whose el~ctrochelnical behauiour in association with rnecliator compounds hauc! b~!~!n stuclied by the ~ppllcants include the FollowincJ:-E.nZyTTlf! SLIbs_ratf!
Flauo--~roteins F'yru~ate Oxidas~! Pyru~at~!s l -~nlino ~cicl Oxidase L-~lnino ~cicls flldf-!hydf-! Oxidase ~'ldeh\/df!s Xanthine Ox i d d S e Xanthinf.~s G~.~ICOSf! r~xiclasf-! Gl.UC-~S~!
Glycollate Oxidase C;lycollate Sarcc,si.ne Oxidasf.! ',arcosine Galactose Oxiclase Galactose Di.aphorase N~DH
(,lutathione Red~lctase N~I~PH
P-~Q Erlz~rTIf!s ......... .................... ... .
Cl~lcosf! [) ~! hyclrogf.~nasf! f,]ucosf!
Methanol Dehydrogenase Methanol ancl other nl kanols Methylamine Dehydrogenase Methylamine C,!LtochrorTIe B--li.nked Enz.ynes .. .. ,_.. _._ .. _ _.__. _ _ __ .__ lactate Oxidase lactate Metal.lofla~ elns Carbon monoxide Carbon ~onoxide Oxidoreductase ~2~3~
It is bellelJed that any of these enzyrne~substrate pairs could be wtillised in association with thc! mc!diator in th~ present in~ell-tion gilJen solne l:irnltatiorls on the assay concditions which would be obuious to the man skilled in -the ar-t. O-f these pairs it is clearly acl~Jantageolls to uti.].ise thosc! c!nzymc!/swbs tra-te pairs whose beha~iour is r.!stabllsllecd ln rnos t detall ancl wlllch 9i~J~! good prr! r r!rably li.nr!ar rc! s pons (I! O~Jr!r thC! r`Xp~! C l:r!cd rneaswrernent range.
Ferrocenr!s (bis- cyclopentadir..!nyl iron and i.ts cleriuatiues) ha~e ad~)antages o~Jer other rned~ tc)rs wsed with enzyme/substrclte rcactlons for charg~ transfer :, p u r po s e s .:
hc! uni.ql~e s tru c -turc! and plAopr rti.c! s c)f f ~!rroc ~!nc! ( b i. s cyclr.lpc!r1-tadi-!nyl lron: FC!C P2 ) and i t s dr!r:i~a ti~J~!s h as r e su] te d in a c onsid er ah].-! amoun L c~f theo r e ti c a 1 a nd experilrlental shldles. First synthesised in 1~51 fe!rrc)cene was the earllr!st exarrlpl~! of the now well- known rn~?.tallocene colnpounds.
Whils t ferrocen-!s had been found to li.mit ~Jalue in spectrophotometric assays as a result of their poor solwbility ~ in aqweows solution and low exti.nction coeffic:lents they ha~Je becn fownd to be rnore swlted to a bio-c!lectrochernlcal systerrl. Ferrocenes hal)e:
--' :
(a) ~ wi~e range of redox potentials accessible through substitution oF -the cyclop~ntadienyl r.ings which can b~ functionali.s~!d, (b) electrochemically revc!rsible one--electron redox properties;
(c) a p~l-ind~!pend~1nt redox pot~Jntial and a slow autoxidation oF the recluced Forln.
Wi.thi.n this g~n~!ral class clf f~!rroc~!n~!s, i.~! th~
rnonolneric or polyrneric deriuates substituted around one or both rings, W~! ha~e found certain indi~ ual ferrocenes such as are llsted below:
rerrc-ene deri.vati~e Fo _ _ Solubl]lt~__ E
l,l'-dlrnethyl- 100 I,D
aceti.c aci.d 1~4 5 370 hycdroxye.thyl- 161 S
f ~! I^ roc~!n ~! 165 ]:,D 335 l,l'bis(hydroxyrnethyl)-- 224 S 385 monocarboxyli.c ~cicl 275 5 420 1,1'-d1carboxyllc acid 285 S
chloro- 345 I,D
methy1 trirnethylamino- 400 S
:: :
: :S indicates were solubllity; I,D means insoluble and :~ : detergent solubilised in 3% Tween-20. E is in rnU ~s standard calom~l elecrode, E is measured in : -; 3~
The EO values of various ferrocerles in phosphate buffer at pH 7.0 given in the above table, span a range oE potentlals, E = 100 to 400 rnv vs SCE. The trend ln E values is ln agree-ment with that expected on the basis of substituent effects. In general electron-donating groups stabilize the positive charge and hence promote oxidation more so than electron withdrawing groups.
Ferrocene derivatives which may be utilised in the method of the present invention are not to be considered as lim-ited to the specific examples listed above, for example, the applicants have determined that ferrocene boronic acid, polymeric ferrocenes, boron tetraferrocene and ethylamine ferrocene exhibit mediator properties furthermore, the derivatives of ferrocene listed above may be further modified by suitable techniques known to the man skilled in the art, such as by treatment with iodine, nitrene or diazo precursors or mercuric chloride.
Our European Patent Application 0078636 published May ll, 1983 describes and claims a sensor electrode composed of electrically conductive material and comprising at least at an ex~ernal surface thereof the combination of an enzyme and a medi-ator compound which transfers electrons to the ~ - 13 -3~
electrocle when the enzyrne is catalytically active, Thc! purpose of such an c!lectrodc! is to dc!tc!ct thc presence of, rneasure the alnount of ancl/or rnonitor -the leuel of one or rnore selected components capable of unclertaking a reaction catalysed by the saicl enzyrne.
Examples of electrode configurations, mediators and uses are g.iven in that patent application.
Ihi.s specification mal<es use of the chemical properties of the rllediat:ors and their derivatlues as exernpli-fied in our earli~!r patent applications.
:
: ~ : rhis :specification also rnakes use oF the electrode :structures and materials disc].osecl i.n the appllcants earl.ler filings, included herein by reference, such as and for exarTIple elc!ctrocle-rrlaterials selected from the ~ : group cornprising; gold, platinul-n, silver, carbon Oln a - one-dimensional conductor.
: :
While the exarnples o-f rnediator cornpounds giuen above are generally li.mited to the ferrocen~ group of mediators, :~ the present inuention may also ernploy the non-ferrocene : ~ ~
:mc!diators. Seueral classes of compounds may pc!rforrrl thc!
~:: funct~ions of a rnediator, that is transfer charge frorn an ~enzyme to an electrode surface (or in cc!rtain cases to a :, ;35~
further enzyrne).
Sys-tems which have been studied ha~e incluclecl t.he Following m~dlator cornpouncls;
Carbon--bororl compouncls (includi.ng the carboranes) ~iologens (N,N'-dialkyl of or d.iaryl deriuatives of 4,4'--bipyridyl) Me~al. carbc)nyls (includi.ng chromium carbonyl) One-Dirnensional Conductors (lncludirlg the salts of TCNQ) Phenazine dyes (including phenazine methosulphate and phenazine ethosulphate), ancl Metal.loporphyrins (i.ncluding cytochrome--C) Howeuer the preferred class o-f mecliators are the transiti.on meta]. complexes par-ti.cularly those in which the Inecllator cornprises a-t least one, and preferably two organic rings, the or each of which is characterised by at least two double bonds that are conjugated and a rnetal atom in electron--sharing contact with each of the rings, since these have stability, exhibit rapid electron transfer reactions, have a wide range o-F redox potentials and are in rnany cases oxygen insensitive.
The preFerred mediators are generally those which have lron, nickel or ruthenium as the transi-tion rnetal.
~lthough the preferable specific bincding reaction in the 3~
method of the present invention is that between avidin (or streptavidin) and biotin, it should be noted that the invention is not to be taken as limited to this specific binding reaction but also extends to the following pairs of specific binding partners;
antigen and antibody, hormone and receptor, lectin and carbohydrate, cofactor and enzyme, and, nucleic acid and complementary nucleic acid.
The preferred method of determination of mediator in solution is by the use of cyclic voltametry as described variously in our above-mentioned patent applications.
It should be noted that the complete cyclic voltammogram need not be taken in every case, and in many instances it is sufficient (as illustrated by example hereafter) ~o polse the cell at a particular potential and to make aIl current and/or voltage and/or time measurements at this potential. As above, the man skilled in the art will be conversant with such techniques and therefore they will not be described in detail herein.
According to a second aspect of the present invention there is provided a nucleic acid sequence probe for 6~
establishing the presence and/or copy number of a giuen sequence in a single--stranded nwcleic acid molecule wherein a probe nucleic acid sequence is linked to;
a) either a rnediator cornpouncl or an enzym~
electrochernically co~lpled by the saicd rnediator cornpouncl, and, b) biotin, wherc!in binding of thc! prol:)c! nuclc!ic acid sequc!ncc! to the target nuc1eic acid sequence rnodifies the electrochemical coupli.ng between the mediator cormpound and the enzyrne ~ccording to a third aspect of the present in~ention there is pro~Jided a rne-thod of assay for nucleic acids wherein an electrode is placed in contact with an enzyrTIe and a substrate oF the enzyrne, and a rnediator cornpound is associat~!d with th~l enzyTne to transfer charge from the enzyrne to the elec trocle as the con~Jersion of subs trate into product proceeds, and, a probe nucleic sequence is linked to either the enzyrne or the rnediator cornpollnd, whc!rc!by th~? acti~i ty thc! c!nzynl~! ancd th~
rnediator cornpound and hence the quantity of charge being transfc!rrQd to the ~ !ctrodfJ is rrlodifi~!d by th~! pr~!senc~!
or abs~nce of a cornplernentary target nucleic acid 3~3 sequence bound to the probe n~clelc acid seguence.
The general comments as to the nature of the mediator, enzyme, methodology and apparatus given in respect of the first aspect of the invention apply equally well to these second and third aspects which should be read accordingly.
The basls of the present invention is that a current passes in response to the presence o an electrochemlcally active o substance or ~mediator~ at an electrode surface, as described in our earlier patent applications, especially our European Patent Application 007863~. The magnitude of the current passed varies either (a~ in proportion to the amount of DNA-probe / target-DNA
complex formed, or (b) as a perturbation of an existing steady state current which is either enhanced or diminished, in propor-tion to the quan-tity of DNA probe / target-DNA complex formed.
In one preferred embodiment of the present invention the electrode exhibits reversible electron transfer with biotin-labled cytochrome-C. The biotin-labled cytochrome-C acts as a mediator compound, but can also bind to avidin as described in general terms above.
.
.
i3~
~ Jidin or Strepta~idin is addc!d tc~ the rf!action rnixture and binds -to the mediator, reducing the transfer of chargf! to the elf!c trode . The adcliti.on of bic tin-lablf-~d nucleic aclcl (DN~ or RN~) to the rnixture sets up a corrlpf!ti ti~Jf! spc!cific bi.nding rc!ac l~.i.on bc!tlllf!f!n on thf! onc hand a E~iotin Cytochrolne-C (first 1:igand-rnediator) corrlplf.!x wi.th al)idin (anti.li.fJand) and on the other hand an a~Jiclin (antiligand) cornplex with the hiotin-labled nucleic aci.d (DNf4 or RNf~) (second li.gand). ~15 the aoidin (or strepta~Jidin) concentration is fixed, an incrf!asf! . in the concentration of the a~idin-biotin-nucl.eic acid corllplex oill cause a corresponcli.nc~ i.ncrf!asf! i.n the concentra-tion of the fr~f!f!
: - tliotin Cytochro!ne C species, which will cause a rela-ted increasf! i.n the rate of transff.!r of charge frc~m thf!
enzylne to the elec-trode surface.
~lthou~h -the abo~Jc~ errlbodirr!f.!nt of thf inlJention has bef!n described in terrns o-f a firs t ligalld 1inked to the mediator, it should be understood that the inlJc!ntion is also en~isdfye.d as being configurecl with the rnediator linkf~d to f!ithf!r thf! f!nzyrrlf! or thf! subs trate .
In a further preferred ernbodirnen t of the pres ent in~ention the elf!ctrodf! surfacc! df!tf!cts and measurf!s an enzyrne catalysed reaction in the presence of a rnediator, which in a morf-7 preferablf.! ernbodirrlf!nt is cytochrome-C
:. :
~Z~j3~
co~alent.ly linl<~d to a nucl~ic acid probe. The electrochernical auailabillity of the rnediator is affected by th~ degrfee of hornologows bincling betwf!f!n the nucleic acld probe and the targ~t DN~.
In a yet furtller pre-fc!rred errlbocli.rrl~!nt of -the present inuention, the electrode det~cts and rneasures, in the preserlcf! oF a mr!cliator, an enzyrrl~!-complf!x catalysed reaction. :Ln a particular ernbodirnent, the enzyme complex comprises a biotin-peroxiclasf!-auidin complf!x which is capable oF binding further biotin or biotin labled nuclf~ic acid. ~ steady--stat~! current is obtained using the peroxide subs-trate. rhiS current decreases when biotin-lablecl nucleic acid i5 added to the assay rnixture .
:
In c!ach of the threfe errlbodirrlents clescri.bed aboue there is a cornpetitiue reaction between the F)robe-containing specif!s and the c~lectrocherrli.cal system comprisirlg the rnediator, enzyrne and substrate. The rate of charge -transfer is peturbed by a lowf.!ring of the a~ailablity of the probf.!~containing species. Consequen-tly, when a sample containing targc!t nucleic acid is added to the assay system, the degree of hornologous binding between ::. the probf~! nlJclf.!ic acicl and thc! targfJt is rf!fl~ctf.!d in ~
lowering of the auailabllity of the free probe containing species, and a corrf~sponcding change in the .
, ,3~
rate of charge transfer.
In a still furth~r prc!ff~rr~!d ~!rllbodi.~lf~!nt of thE! pr~sfJnt invention, the tnediator .is a ru-theniuln cornpound The rat~ of r~!clucti.on of the ruth~!niurn corrlpouncl p~nta--alnlnine-isoni.cotinarlline--rutheniuln (I:rI) was First notr!cl to be high in 1~70 by raube~ Since this ti.me a quantity of work has been done with ru-theniuln compourlds both in liqui.i systf.!rrls ~ncl on ~!lf.!ctrod~!s, how~!v~!r th~!
incorporation of a ru-theniuln compound into a charge transfc!r c!l~!ctrodc! is pr~!sc!ntc!d llc!l~! for th~! first tirrlc!
~Sp~ci~ic ruthen1.uln cornpounds which are preferr~d in : c!rrl~odimc!nts of the prc!sc!n-t in~c!nti.on inc].udc! thc :
: : following;
I:Ru(NH~)5py]
CRU(N~13)6J
1: RU ( N~l 3 ) 5 L ]
Wherf! L is 4-aminomethylpyridine (~MP), or L is N--(4--plc~olinic)-benzamide (PB~
[Ru(NH~)50H2] ' , and, ; [Ru(NH3)5~1]3~'2+
m~thod for attaching the first abo~f~! listf.!d compound to a gràphite electrode is disclosed by Ko~al and ~nson ,' 3K~
(as re-ferenced aboue).
It is also enuisaged that -the present inuention extencls to nouel conjugates oF DN~ or RN~ and rutheniurn cleriuati.ues. It has been known [Clarke and Taube Journal of the ~rnerican Chernical Society 97;6 March 19 t975] to prepare adducts of rutheniuTn compounds with xanthines, and lt is therefore beliued possible that the m~!thocis of the present in~ention may be extende~ to nucleotide derivatiues which are rutheniurn adducts.
In orcler that the present inuention may be further uTlderstood, it will be hlrther illustratecl by way o-f exampl~ wi.th reference to -the accompanying figures where1n;
Fiqure_l; shows an assay protocol in which the nucleic acid is bound to -the enzyrne, Fl~ur~ 2; shows an assay in which the nucleic acid is bound to the rnediator, F~iqu e 3; shows an assay in which the nucleic acid lS bound: to a linker which has an aFfinity for the : ~ elctrode surface, : ~ig~ _4a; shows the cyclic ~oltarnrnogram of 2.8mg `. ,~
biotin cytochroTnf3--C in thc! prc!sf.!ncf! of horsc!radish peroxidase .
Flclure_ 4b; shows -thf.! samf! as 4a on additi.c)n of 300urn Hydrogen peroxide, F ~ ,1rf! S; shows a Y"r rf!ccJrc1f.!r trclce of' thf.! C;urrf~ t produced in the prf!ser1ce c)f 1, 41r1g/1r1l of~
Ibiotin- cytochrorr1c! C:, :L05L1g/rr1l horserc1c1i.sh peroxidase, at a BPS surface 1noc11~ !c1 gol.d workinq elc!ctrodf!, I -lgUrf! __",,6; shows thf! cycli.c uol-tamrr1ograrr1 of [R1l(N~13) 5]~)y , wi.t~l golcl hS .:~ worki.rlg f!]f!C trodE`
i.n t1~f! prr!sf!ncf.,! of 6uM bi.c) t:i. rl- pfJr~ x~l.dasf! dui,di,n f.!rrlzyrr cornplf3x, -lgur~.~ 7; shows f'ur-thf.~r c1f tai.1s of' f i.gurc! 7, anc1, ;Fi,c,~ure_8, shows a Yt plc)t c,f th~-~ catalytic current against timc~ with thf! additl.on of' L)iotinylatf!d L)Ni~ to the assay r1lixture.
~ ' Exampl f! l; ENZ`~ME ~ CWEL~ 10 DN~ PROBF
In: Figure l, E is an enzyrne, for example, glucose oxidast3, which c~talysc!s thc! conuc!rsi-Jrl of a subs trat.t.! X
: ~ :
~; (for exa:rnpl~ glucose) to a procluct, P, (for exa1nple gluconic acid) and libf~!ratf`s fJlf-!ctrons, f~!, which r~!duc~
~ , ~
" ~
7he.r(:! i.s a particu].ar need in may areas of biocherrlical reseach alld collllrlerce to be able to assay for the pr-esence of nucl.eic acids i.n a salnple and further to assay for the (?xist~nce of particular sequences within t ~ l i. CI ~ c ~ c l C i. Cl s .
Ih~! c!xp].oi.t.i~Liorl of t:hc! hybri.ti i sa ti.-Jn bc!Lwc!c!n t.wo single stranded DN~ or RN~ rr~ lecull?s wh:ich haue a corrlpl.errlc!rltilry sc!cluence has be~n on~! approach to this probleln. ~olecules for use in such assays which are capahle of binding to a DN~ or RN~ sequence which is def.ined l:o a particlJlar le~el of holnology are known as DN~-probes.
I<nown ~N~ (RNn) probe Lechiques share a simllari-ty in thi~t the DN~ (RNn) polyrner is not readily detectable by its i.nher~nt biocherl)ical acti~i.ty. It is therc!~ore ~q necessary to mark the polymer with some signal-producing chernical or biochernical species, such rnethods as are presently known include the following prior ~rt:--PRIOR ~ Rl h~ldln-Biotin_ R~actlon; this technology reli.es ~n the aFf~inity of the egg-whltc! glycoprotein a~idin for bi.o-ti.n. Biotin (Uitamin ~1) can be coval~!ntly linked to the rucleotide residuc!s which cornprise the rnonolneric subunits oF the r~Nh polymer. The modified subunits can still undergo the classical binding reaction between complementary strands of double~stranded DNh, and thus can be incorporated into syn-thetic DNh probes. ~t pr~!s~!nt, to clf~t-!ct the pr~!s~!nce of SUC~I probes which ha~e forrned short double-stranded regions af-ter exposure to comp].errlentary sample DNh, the unbo~rid probe must Firs-t be separated frorn the sarnple I~N~/bound probe complex. rhis is norrnally done by perforrning the binding reaction in conditions under which the salnple DNh is immobi].ized on a substrate and washing, although cen-trifugation rnay perform the sarne function. The bound probe is dete-cted by -the addition of a~idin to which either a fluorescent- rnarker~labeled antibody or an enzyme has been attached.
one problem with the above method is that small . ,;
J,3~
oligonucleotide probes (20 nucleotides) contain only a small number of biotinylated sites, limiting the amount of avidin which can be bound. Attempts have been made9 with some success, to add long "tails" of up to several thousand bases to the probe DNA, in which case only the tail need be labeled. The method can detect up to a resolution of 10 139 of DNA, or about 105 copies of a single gene. Although originally the marker on the avidin was horse radish peroxidase (a rather short-lived enzyme) the method has now been extended toinclude alkaline phosphatase. Unfortunately the method is generally difficult to establish for a new diagnostic scheme as either the biotin-linked probe DNA is difficult to prepare to the labeled tail interfers with sensitivity.
USE OF MEDIATORS:
Genetics Internationals' Canadian Patent Application 453584 of 4th May 1984 discloses a method in which at least one of a mediator and an enzyme are chemically linked to a nucleic acid probe sequence whereby speciflc binding of the probe sequence to the tarqet sequence in a single-strand nucleic acid material to be availability of the chemically linked species as detected by a sensor electrode in presence of` the enzyme substrate, whereby thepresence of the target sequence 3~
can b~ dett~ctecl ~s di.sclosed in the spc!cification the nuclei.c acid sequence can be RNh e.g messergt~r RN~ but is usually DN~.
sui.tablt! rnethod for the practice of thi.s techniqut-! has been giuen as follows;
(a) pro~iding a singlc! strand nucleic acid material to be in~estigated for a gi~en target sequence;
(b)s~:!].~cting a probc! rrlatc!ri~l with cl st!quc!ncc of nuclt!ic ac.ids cornplelnentary to the target seque~nce;
(c) choosi.rlg a proced~lr(! frorn ~mong;
.
(i) chelnically linking the probe with an enzyln~ and acldi.ng the enzyrrl~ linked probe to a solution containing both a substrate For the enzyrnt~ and a rnediator (ii) chemica]ly linkillg the probc! with a mediator and adding the mediator-linked probe to a solution containing both a substrat~ and an enzymt.! For the saicl substrate;
(iii) chemically linking thc! probe with a mccli.ator/
, 3~
enzyn1rl combination and adding the so-modified probe to a solution containing a substrate for the enzyrne (cl) contacting the solution containing the chemically linkecl probe sequence with a sensor electrocle whereby chargc! is transfc!rrr!d hy tl~r! mr!di.ator to thr! r-Jlr!ctrodr~
Froln the enzyrne-catalysed substrate reac-tion and (e) contacti.ng the soluti.on with the sirigle strandr!d mat~rial, whereby alteration in the a)nount of chargr.! transfr.!rred i5 an lndication of a specific binding reaction between thr! probr! and targr!t affr!c-t.ing thr.! a~ail~bi].ity of enzyrne rnediator or cornbination.
Ihe probr-! matr.!rial can br.! a naturally occuring [)~n fraglnent or a synthetically produced ~rlaterial.
~lterati.ons i.n the sequence of s-tr!ps can bre readily en~isaged. ~lso the sensor electrode itself can include the mediator or the r!nzyrne although genr.!ra].ly it is pre-ferred for the probe sequence anrd the tar~qc!t sequence botll to bc! prr!sc!r~t i.n sol~ltion.
rhe rnediator can be linl<ecl indlrectly to the probe sr-!quencr.! by a ].i.nker group and a nlaterial reacti~e to 3~
thc! ~.inker groups can b~! pr~!s~!r~t on th~! ~!].c!ctrod~! In this case the whole cornplex is present on the electrode;
when a target sequence is present, and binds to the probe sequence, an altera-tion in elec-trode current is produ c ed .
ME[)Ih~OR~ _)ISCLOJE[~, Th~! U S~! of rut~l-!ni.~lTrl corrlpl-!xes on a graphitc! ~!lc!c-trc)dc is cdisclosed in l<o~al and hnson [~naly-tical Chernistry, Uol 50 No 2, Fc~!b 1978 pg 223~. In which the follooing ruthenl.uln cornp~lexes are cliscussed;
Ru(NH )3-~2-~
Ru(NH3)5py Ru(NH3)5L
Wher~e L i5 pyridine, 4 aminorrlethylpyridine (hMP), or L
is N-(4 pico1inic)~-benzamide (PBh) -Ru(NH3)50~2 ' Ru(NH3)5~13~'2~
E RORS OF METh OLISM
':.,' 3~
One airn of the DN~ probe technology ancl the enzyrne dc!tection/assay tc!chnology so fal~ dc!~c!].opc!d has bc!c!n to detect inbuilt errors of rnetabolisrn which lead to a uai^lc!ty of ~I y~!nC!ti.C Cli,sC!clS ~!S and i.rlhc!rittlb]c!
disorders. ~rnong such clisorders arc-l: farnilial Goiter (i.odc)tyrosirlt! dc!ha1f~g~!nclsc! clc!fc!ctivc!) Maplc! syrup ul^inc disease (alpha--l<eto clecarboxylase deFective) Xanthinuria (Xanthine oxidase defective) and Me-th~3lnoglobinelrlia (Methelnoglobin reductase defestive).
f~l11 list of 3500 conditions duc! to d~!fc!ctivF.! gc!nc!s can be founcl in McKusick's "Menclelian Inheritance in Man".
Onc! partic~l].ar disc!asc! of i.ntc!rc!st is sicklc!-cc!ll amelnia in which one of the norlnal Globin qene coclons corl^esponcling -to the f:ifth sixth and seventh arrlino ac:ids of a particular chain are not as norlnal CC~-C~G-G~G but read Gcr--G-rG-c~G/ thereby rep1acing valine for glutarnic acid at position six. 5yn-thetic o].i.gonuclc!oti.d~!s ha~c! bc!c!n rrladc! which can act as probc~s For the sequence ~aria-tion in prena-tal diagnosis of the disc!asc! and succc!ssfully distinguish bc!tlAJc!c!n t~le wild type and the aberant gene. Assay of the colnponents of the binding rni.xture was perForrlled by a radiot,lc tiue tracer rnethod ~ sirnilar approach has been tal<en with the point mutation reportecl to cause antitrypsin ~, ;3~
deficiency.
~s can be seen frorn the aboue, howeuf!r, the methods of cletailed rnetabolic assay presen-tly auailabl~ haue certain disacluantagf!s i.n that they requirfe somf! or a].l of the followlrlq; short lifetirne reagf!nts (e1ther l~adi.oactiuf.!, ai~ s~!rlsiti.uf! or~ l.i.yht-sf.!llsitiuf!)~ lligh1y trained staff or (for autoradiography, ESR rnf!asurelnents or lc)w-~lf.!ue]. 1i.gllt clf!tf~c-ti.on) f!xpf~nsi.uf-! f-!qui.pmf!nt.
Known rne.-thods of assay -for binding reactions other than antigf~n/ant.i.bo(:ly and DN~/cornplf:!rrlf~ntary DN~ are genf!rally sirllilar tc) the aboue rne-tllods and s~lffer -Frorn simlldr prob].f!ms, ~ccorclirlg to onf-~ aspfec-t c,:f the prf!sf!nt inufenti.on thferf!
ls prouldfed an assay for n~lcll?ic acid which cornprises tll f ! C; t f ! p S C~ f;
(a) proui.ding a probe matf?ria]. corrlpri.si.ng;
(i) a sequence o-f nucleic acids cornplernentary to a giuen targfet sequence and, (ii) a fi.rst li.gand chc-!trliccll].y li.nked therf~to and capable of a specific blndinq reaction with an arti].igand;
(b) cont~cting the said probe matf!rial with an assay ;3~
systenn cornprising;
(i) a suitable mediator enzymc! suhstrate SySt.eTn capablfs of transferring charge to an tslectrode surface when t.he enzymt.! is catalyti.caly acti~e and;
(ii)a sfscond ligand chern.ically linked to one o,'- said TTIediator f.!nzyTTIf! or substratf! whc!rf!in thf! st~!cond liyand is capab1e o-f a competitiue binding reaction witll -the antil]gand and;
(ii.i) the said antiligand whereby the saicl first ligand cornpett-.!s with thf! sai.d second l.igar)cl in a specific binding reaction with -the antili.gancl and;
(c) contacti.ng thf..! abo~e systenn wi.th a soluti.on suspected of containing -the sa:icd target sf!quence whereby the binding of any of the saicl target sequt!nct.! prt.!st.!nt to the probe a-ffects the a~ailiabil~Lity of the First ligand and therefort.! alters thf! rate of charge transfer to the electrode.
By elTIploying a mt-!thod as described abo~e i-t is possi.ble to perforlrl the assay with an alnpl.iflcation step thereby i.ncreasing the resoluti.on of the assay.
Furtherlnore the rnethod of the present in~ention dcles not require the short li~c-!d radioactive assay components which are ernployed in othfsr types of assay.
;3t?3~
Enzyrne~substrate pairs whose el~ctrochelnical behauiour in association with rnecliator compounds hauc! b~!~!n stuclied by the ~ppllcants include the FollowincJ:-E.nZyTTlf! SLIbs_ratf!
Flauo--~roteins F'yru~ate Oxidas~! Pyru~at~!s l -~nlino ~cicl Oxidase L-~lnino ~cicls flldf-!hydf-! Oxidase ~'ldeh\/df!s Xanthine Ox i d d S e Xanthinf.~s G~.~ICOSf! r~xiclasf-! Gl.UC-~S~!
Glycollate Oxidase C;lycollate Sarcc,si.ne Oxidasf.! ',arcosine Galactose Oxiclase Galactose Di.aphorase N~DH
(,lutathione Red~lctase N~I~PH
P-~Q Erlz~rTIf!s ......... .................... ... .
Cl~lcosf! [) ~! hyclrogf.~nasf! f,]ucosf!
Methanol Dehydrogenase Methanol ancl other nl kanols Methylamine Dehydrogenase Methylamine C,!LtochrorTIe B--li.nked Enz.ynes .. .. ,_.. _._ .. _ _.__. _ _ __ .__ lactate Oxidase lactate Metal.lofla~ elns Carbon monoxide Carbon ~onoxide Oxidoreductase ~2~3~
It is bellelJed that any of these enzyrne~substrate pairs could be wtillised in association with thc! mc!diator in th~ present in~ell-tion gilJen solne l:irnltatiorls on the assay concditions which would be obuious to the man skilled in -the ar-t. O-f these pairs it is clearly acl~Jantageolls to uti.].ise thosc! c!nzymc!/swbs tra-te pairs whose beha~iour is r.!stabllsllecd ln rnos t detall ancl wlllch 9i~J~! good prr! r r!rably li.nr!ar rc! s pons (I! O~Jr!r thC! r`Xp~! C l:r!cd rneaswrernent range.
Ferrocenr!s (bis- cyclopentadir..!nyl iron and i.ts cleriuatiues) ha~e ad~)antages o~Jer other rned~ tc)rs wsed with enzyme/substrclte rcactlons for charg~ transfer :, p u r po s e s .:
hc! uni.ql~e s tru c -turc! and plAopr rti.c! s c)f f ~!rroc ~!nc! ( b i. s cyclr.lpc!r1-tadi-!nyl lron: FC!C P2 ) and i t s dr!r:i~a ti~J~!s h as r e su] te d in a c onsid er ah].-! amoun L c~f theo r e ti c a 1 a nd experilrlental shldles. First synthesised in 1~51 fe!rrc)cene was the earllr!st exarrlpl~! of the now well- known rn~?.tallocene colnpounds.
Whils t ferrocen-!s had been found to li.mit ~Jalue in spectrophotometric assays as a result of their poor solwbility ~ in aqweows solution and low exti.nction coeffic:lents they ha~Je becn fownd to be rnore swlted to a bio-c!lectrochernlcal systerrl. Ferrocenes hal)e:
--' :
(a) ~ wi~e range of redox potentials accessible through substitution oF -the cyclop~ntadienyl r.ings which can b~ functionali.s~!d, (b) electrochemically revc!rsible one--electron redox properties;
(c) a p~l-ind~!pend~1nt redox pot~Jntial and a slow autoxidation oF the recluced Forln.
Wi.thi.n this g~n~!ral class clf f~!rroc~!n~!s, i.~! th~
rnonolneric or polyrneric deriuates substituted around one or both rings, W~! ha~e found certain indi~ ual ferrocenes such as are llsted below:
rerrc-ene deri.vati~e Fo _ _ Solubl]lt~__ E
l,l'-dlrnethyl- 100 I,D
aceti.c aci.d 1~4 5 370 hycdroxye.thyl- 161 S
f ~! I^ roc~!n ~! 165 ]:,D 335 l,l'bis(hydroxyrnethyl)-- 224 S 385 monocarboxyli.c ~cicl 275 5 420 1,1'-d1carboxyllc acid 285 S
chloro- 345 I,D
methy1 trirnethylamino- 400 S
:: :
: :S indicates were solubllity; I,D means insoluble and :~ : detergent solubilised in 3% Tween-20. E is in rnU ~s standard calom~l elecrode, E is measured in : -; 3~
The EO values of various ferrocerles in phosphate buffer at pH 7.0 given in the above table, span a range oE potentlals, E = 100 to 400 rnv vs SCE. The trend ln E values is ln agree-ment with that expected on the basis of substituent effects. In general electron-donating groups stabilize the positive charge and hence promote oxidation more so than electron withdrawing groups.
Ferrocene derivatives which may be utilised in the method of the present invention are not to be considered as lim-ited to the specific examples listed above, for example, the applicants have determined that ferrocene boronic acid, polymeric ferrocenes, boron tetraferrocene and ethylamine ferrocene exhibit mediator properties furthermore, the derivatives of ferrocene listed above may be further modified by suitable techniques known to the man skilled in the art, such as by treatment with iodine, nitrene or diazo precursors or mercuric chloride.
Our European Patent Application 0078636 published May ll, 1983 describes and claims a sensor electrode composed of electrically conductive material and comprising at least at an ex~ernal surface thereof the combination of an enzyme and a medi-ator compound which transfers electrons to the ~ - 13 -3~
electrocle when the enzyrne is catalytically active, Thc! purpose of such an c!lectrodc! is to dc!tc!ct thc presence of, rneasure the alnount of ancl/or rnonitor -the leuel of one or rnore selected components capable of unclertaking a reaction catalysed by the saicl enzyrne.
Examples of electrode configurations, mediators and uses are g.iven in that patent application.
Ihi.s specification mal<es use of the chemical properties of the rllediat:ors and their derivatlues as exernpli-fied in our earli~!r patent applications.
:
: ~ : rhis :specification also rnakes use oF the electrode :structures and materials disc].osecl i.n the appllcants earl.ler filings, included herein by reference, such as and for exarTIple elc!ctrocle-rrlaterials selected from the ~ : group cornprising; gold, platinul-n, silver, carbon Oln a - one-dimensional conductor.
: :
While the exarnples o-f rnediator cornpounds giuen above are generally li.mited to the ferrocen~ group of mediators, :~ the present inuention may also ernploy the non-ferrocene : ~ ~
:mc!diators. Seueral classes of compounds may pc!rforrrl thc!
~:: funct~ions of a rnediator, that is transfer charge frorn an ~enzyme to an electrode surface (or in cc!rtain cases to a :, ;35~
further enzyrne).
Sys-tems which have been studied ha~e incluclecl t.he Following m~dlator cornpouncls;
Carbon--bororl compouncls (includi.ng the carboranes) ~iologens (N,N'-dialkyl of or d.iaryl deriuatives of 4,4'--bipyridyl) Me~al. carbc)nyls (includi.ng chromium carbonyl) One-Dirnensional Conductors (lncludirlg the salts of TCNQ) Phenazine dyes (including phenazine methosulphate and phenazine ethosulphate), ancl Metal.loporphyrins (i.ncluding cytochrome--C) Howeuer the preferred class o-f mecliators are the transiti.on meta]. complexes par-ti.cularly those in which the Inecllator cornprises a-t least one, and preferably two organic rings, the or each of which is characterised by at least two double bonds that are conjugated and a rnetal atom in electron--sharing contact with each of the rings, since these have stability, exhibit rapid electron transfer reactions, have a wide range o-F redox potentials and are in rnany cases oxygen insensitive.
The preFerred mediators are generally those which have lron, nickel or ruthenium as the transi-tion rnetal.
~lthough the preferable specific bincding reaction in the 3~
method of the present invention is that between avidin (or streptavidin) and biotin, it should be noted that the invention is not to be taken as limited to this specific binding reaction but also extends to the following pairs of specific binding partners;
antigen and antibody, hormone and receptor, lectin and carbohydrate, cofactor and enzyme, and, nucleic acid and complementary nucleic acid.
The preferred method of determination of mediator in solution is by the use of cyclic voltametry as described variously in our above-mentioned patent applications.
It should be noted that the complete cyclic voltammogram need not be taken in every case, and in many instances it is sufficient (as illustrated by example hereafter) ~o polse the cell at a particular potential and to make aIl current and/or voltage and/or time measurements at this potential. As above, the man skilled in the art will be conversant with such techniques and therefore they will not be described in detail herein.
According to a second aspect of the present invention there is provided a nucleic acid sequence probe for 6~
establishing the presence and/or copy number of a giuen sequence in a single--stranded nwcleic acid molecule wherein a probe nucleic acid sequence is linked to;
a) either a rnediator cornpouncl or an enzym~
electrochernically co~lpled by the saicd rnediator cornpouncl, and, b) biotin, wherc!in binding of thc! prol:)c! nuclc!ic acid sequc!ncc! to the target nuc1eic acid sequence rnodifies the electrochemical coupli.ng between the mediator cormpound and the enzyrne ~ccording to a third aspect of the present in~ention there is pro~Jided a rne-thod of assay for nucleic acids wherein an electrode is placed in contact with an enzyrTIe and a substrate oF the enzyrne, and a rnediator cornpound is associat~!d with th~l enzyTne to transfer charge from the enzyrne to the elec trocle as the con~Jersion of subs trate into product proceeds, and, a probe nucleic sequence is linked to either the enzyrne or the rnediator cornpollnd, whc!rc!by th~? acti~i ty thc! c!nzynl~! ancd th~
rnediator cornpound and hence the quantity of charge being transfc!rrQd to the ~ !ctrodfJ is rrlodifi~!d by th~! pr~!senc~!
or abs~nce of a cornplernentary target nucleic acid 3~3 sequence bound to the probe n~clelc acid seguence.
The general comments as to the nature of the mediator, enzyme, methodology and apparatus given in respect of the first aspect of the invention apply equally well to these second and third aspects which should be read accordingly.
The basls of the present invention is that a current passes in response to the presence o an electrochemlcally active o substance or ~mediator~ at an electrode surface, as described in our earlier patent applications, especially our European Patent Application 007863~. The magnitude of the current passed varies either (a~ in proportion to the amount of DNA-probe / target-DNA
complex formed, or (b) as a perturbation of an existing steady state current which is either enhanced or diminished, in propor-tion to the quan-tity of DNA probe / target-DNA complex formed.
In one preferred embodiment of the present invention the electrode exhibits reversible electron transfer with biotin-labled cytochrome-C. The biotin-labled cytochrome-C acts as a mediator compound, but can also bind to avidin as described in general terms above.
.
.
i3~
~ Jidin or Strepta~idin is addc!d tc~ the rf!action rnixture and binds -to the mediator, reducing the transfer of chargf! to the elf!c trode . The adcliti.on of bic tin-lablf-~d nucleic aclcl (DN~ or RN~) to the rnixture sets up a corrlpf!ti ti~Jf! spc!cific bi.nding rc!ac l~.i.on bc!tlllf!f!n on thf! onc hand a E~iotin Cytochrolne-C (first 1:igand-rnediator) corrlplf.!x wi.th al)idin (anti.li.fJand) and on the other hand an a~Jiclin (antiligand) cornplex with the hiotin-labled nucleic aci.d (DNf4 or RNf~) (second li.gand). ~15 the aoidin (or strepta~Jidin) concentration is fixed, an incrf!asf! . in the concentration of the a~idin-biotin-nucl.eic acid corllplex oill cause a corresponcli.nc~ i.ncrf!asf! i.n the concentra-tion of the fr~f!f!
: - tliotin Cytochro!ne C species, which will cause a rela-ted increasf! i.n the rate of transff.!r of charge frc~m thf!
enzylne to the elec-trode surface.
~lthou~h -the abo~Jc~ errlbodirr!f.!nt of thf inlJention has bef!n described in terrns o-f a firs t ligalld 1inked to the mediator, it should be understood that the inlJc!ntion is also en~isdfye.d as being configurecl with the rnediator linkf~d to f!ithf!r thf! f!nzyrrlf! or thf! subs trate .
In a further preferred ernbodirnen t of the pres ent in~ention the elf!ctrodf! surfacc! df!tf!cts and measurf!s an enzyrne catalysed reaction in the presence of a rnediator, which in a morf-7 preferablf.! ernbodirrlf!nt is cytochrome-C
:. :
~Z~j3~
co~alent.ly linl<~d to a nucl~ic acid probe. The electrochernical auailabillity of the rnediator is affected by th~ degrfee of hornologows bincling betwf!f!n the nucleic acld probe and the targ~t DN~.
In a yet furtller pre-fc!rred errlbocli.rrl~!nt of -the present inuention, the electrode det~cts and rneasures, in the preserlcf! oF a mr!cliator, an enzyrrl~!-complf!x catalysed reaction. :Ln a particular ernbodirnent, the enzyme complex comprises a biotin-peroxiclasf!-auidin complf!x which is capable oF binding further biotin or biotin labled nuclf~ic acid. ~ steady--stat~! current is obtained using the peroxide subs-trate. rhiS current decreases when biotin-lablecl nucleic acid i5 added to the assay rnixture .
:
In c!ach of the threfe errlbodirrlents clescri.bed aboue there is a cornpetitiue reaction between the F)robe-containing specif!s and the c~lectrocherrli.cal system comprisirlg the rnediator, enzyrne and substrate. The rate of charge -transfer is peturbed by a lowf.!ring of the a~ailablity of the probf.!~containing species. Consequen-tly, when a sample containing targc!t nucleic acid is added to the assay system, the degree of hornologous binding between ::. the probf~! nlJclf.!ic acicl and thc! targfJt is rf!fl~ctf.!d in ~
lowering of the auailabllity of the free probe containing species, and a corrf~sponcding change in the .
, ,3~
rate of charge transfer.
In a still furth~r prc!ff~rr~!d ~!rllbodi.~lf~!nt of thE! pr~sfJnt invention, the tnediator .is a ru-theniuln cornpound The rat~ of r~!clucti.on of the ruth~!niurn corrlpouncl p~nta--alnlnine-isoni.cotinarlline--rutheniuln (I:rI) was First notr!cl to be high in 1~70 by raube~ Since this ti.me a quantity of work has been done with ru-theniuln compourlds both in liqui.i systf.!rrls ~ncl on ~!lf.!ctrod~!s, how~!v~!r th~!
incorporation of a ru-theniuln compound into a charge transfc!r c!l~!ctrodc! is pr~!sc!ntc!d llc!l~! for th~! first tirrlc!
~Sp~ci~ic ruthen1.uln cornpounds which are preferr~d in : c!rrl~odimc!nts of the prc!sc!n-t in~c!nti.on inc].udc! thc :
: : following;
I:Ru(NH~)5py]
CRU(N~13)6J
1: RU ( N~l 3 ) 5 L ]
Wherf! L is 4-aminomethylpyridine (~MP), or L is N--(4--plc~olinic)-benzamide (PB~
[Ru(NH~)50H2] ' , and, ; [Ru(NH3)5~1]3~'2+
m~thod for attaching the first abo~f~! listf.!d compound to a gràphite electrode is disclosed by Ko~al and ~nson ,' 3K~
(as re-ferenced aboue).
It is also enuisaged that -the present inuention extencls to nouel conjugates oF DN~ or RN~ and rutheniurn cleriuati.ues. It has been known [Clarke and Taube Journal of the ~rnerican Chernical Society 97;6 March 19 t975] to prepare adducts of rutheniuTn compounds with xanthines, and lt is therefore beliued possible that the m~!thocis of the present in~ention may be extende~ to nucleotide derivatiues which are rutheniurn adducts.
In orcler that the present inuention may be further uTlderstood, it will be hlrther illustratecl by way o-f exampl~ wi.th reference to -the accompanying figures where1n;
Fiqure_l; shows an assay protocol in which the nucleic acid is bound to -the enzyrne, Fl~ur~ 2; shows an assay in which the nucleic acid is bound to the rnediator, F~iqu e 3; shows an assay in which the nucleic acid lS bound: to a linker which has an aFfinity for the : ~ elctrode surface, : ~ig~ _4a; shows the cyclic ~oltarnrnogram of 2.8mg `. ,~
biotin cytochroTnf3--C in thc! prc!sf.!ncf! of horsc!radish peroxidase .
Flclure_ 4b; shows -thf.! samf! as 4a on additi.c)n of 300urn Hydrogen peroxide, F ~ ,1rf! S; shows a Y"r rf!ccJrc1f.!r trclce of' thf.! C;urrf~ t produced in the prf!ser1ce c)f 1, 41r1g/1r1l of~
Ibiotin- cytochrorr1c! C:, :L05L1g/rr1l horserc1c1i.sh peroxidase, at a BPS surface 1noc11~ !c1 gol.d workinq elc!ctrodf!, I -lgUrf! __",,6; shows thf! cycli.c uol-tamrr1ograrr1 of [R1l(N~13) 5]~)y , wi.t~l golcl hS .:~ worki.rlg f!]f!C trodE`
i.n t1~f! prr!sf!ncf.,! of 6uM bi.c) t:i. rl- pfJr~ x~l.dasf! dui,di,n f.!rrlzyrr cornplf3x, -lgur~.~ 7; shows f'ur-thf.~r c1f tai.1s of' f i.gurc! 7, anc1, ;Fi,c,~ure_8, shows a Yt plc)t c,f th~-~ catalytic current against timc~ with thf! additl.on of' L)iotinylatf!d L)Ni~ to the assay r1lixture.
~ ' Exampl f! l; ENZ`~ME ~ CWEL~ 10 DN~ PROBF
In: Figure l, E is an enzyrne, for example, glucose oxidast3, which c~talysc!s thc! conuc!rsi-Jrl of a subs trat.t.! X
: ~ :
~; (for exa:rnpl~ glucose) to a procluct, P, (for exa1nple gluconic acid) and libf~!ratf`s fJlf-!ctrons, f~!, which r~!duc~
~ , ~
" ~
2~
the oxiclised forrn of a mc!di.ator MoX (For ~!xample the ferriciniurn ion) to the reduced forrn Mr,!d (for exarnple ferrocence.). MrfCl is oxidised at the electrode G the current passed is rneasured and is proportional to the amount of subst.ratf! S presc!nt.
Bound to the enzyrne E by any sui-table rnethod ls a DN~
fragrrlf!nt D which may be c!ither deri~Jf.!d frorrl a naturally occuring DN~ sequence or rnay be synthetic. In the presencfe of an excf.!ss of the substratc! S a steady sta-te curren-t is obtained. ~ sarnple of DN~ which is to be assayed fc~r a parti.culclr -target sfequferlcf? complc!rrlentary to the sequence of the DN~ -fragrnr~ t D is con~ertecl to singl.e strands by any sui.table mf!thnd and then addf!d to the reaction rllixture.
Il: a targc!t sf!quf!ncE! cornplf!rrlf!ntary to the DN~ fragmf!nt D
is present in the rnixture it will b:ind to the fragrnent D and i.nhibit the enzyrrlatic rf!acti.on of the enzyrrlf! E
with the substrate S. Consequently the throughput of substrate S to product P will be reduced and the coupled reduction of Mox will be dirninished. The change in -the rate of reduction of M is reflf.!ctf!d in a rc!ducti.on o ;~
: of the current at the electrode G.
:Thr-! change i.n current is proportional to the arnount of the fragrnented DN~ D which is now bouncl -to target DN~
;..
and hence to thr* arnount of targr.!t DN~ presc!nt Examplc! 2; MEDI~TOR ~T~CHED TO DNQ PROBE
In exarnple B, the redox-ac tive rnediator M (in this exarrlplr.! a sllbsti. tut~!d fc!rroc~!nr!) is attachc!d dirc!c tly (as shown ln Figure 2) to the fragrnented DNh which is -to be used as a probe. The formation of mr!cliator-].inkecl DN~-probe cloes not impede eithrer -the arnperolne-tric respc)nsr! of -the medi.a-tor M nor l:.he b.inding interaction o-f the media-tor-DNh probe wi th the cornplernentar y target sequr!nce containr!d in -the DNQ which i.s being assayecl.
I he alnperornetric response cawsecl hy the addition of the medi.ator-DN~ probe to -the assay mi.xturr! is meas~rr-!d. If genetic rnaterial in a single- strandecl -forrn to which the probc! i s corrlplr!mr!ntary i s prr! s r!n t t~lr pl obr! bi nds to the cornplernentary sequence in the sample DN~. rhis great].y reducr-!s or~ completely inhibits the ampr!rc)rrle-tric response that is the complex of the rn(?diator-DN~ probe and t:he target DN~ is not arnperoTrletrically activr-!. The reduction in the initial arnperornetric response is in direct proportion to the amount of mr!diator-DN~
probe.itarget DN~ cornplex Forrned and- hence to the arnount of qenr!tic material containing a seqllr!nce cornplerrlentary to the known sequence of the rnediator-DN~ probe.
.... .
36;3~
_x_nple 3; MEDI~TOR-~Nr~_ INKER_~"Lr~ ED TO DN~_PROBE
In this r!xample (as shoon in Figurc! 3) the mr!diator-DNQ
probe also contains one or rnore linker groups L., (blotin n~ay for example be used).
rhe rnediator-linker-r~NQ probe is in this exernplary TTlr!thod trr!atr?d with the sarTlplr! suspc!c-tr!d of containing the single strancled genetic rnaterial which is the target for the DNQ probe ancl -the mr!diator--linkr!r-DNQ probe binds to any cornplelnentary sequence present, :
: ~n r;!'lect.rodr! G on whosr! sur-Face is present an elec-trochemical.ly acti~e rllaterial R, that recognises the li.nker group L, for exarrlplr! aui~in labellr!d witl ferrocene, is then irlllnersr!cl in -the reaction mixture.
The curren-t rrleas.lred on applying a potenti.al is re~uced on the binding of the electrochernically actiue rnaterial R to -the nnecliator-linker-DNQ probe /target-DN~ complex.
The recluction of the current is again ln propor-tion to the ~Tnount of th~! added singl~ strandr!d sc!qur!ncr! with a target sequence cornplernentary to the known sequence of the r~NQ-probe.
EXQMPLE _~ SYSTEM COMPRIJING ,BIOTIN C'~TOCHROME--C QND
P,,EROXIDQSE
In this particular ernbodirnent, the electrocle exh.ibits rc!~c!rsiblc! c!l~ctron transfc!r with biotin-labl.c!d cytochrorne-C. -rhe biotin-labled cytochrorne-C acts as a mediator compound, but can ~lsc) t~ind to a~idin as described in general terrns aboue.
The adcli.ti.on of biotin--labled nuc].eic acid ([)N~ or ~N~) to the rrlixture sets up a cornpeti-ti~e speci-fic binding rc!ac-ti.c)n bf!twc!c!n on thc! ~nc! hancl a Biotin Gytochrorr1c!--C
complex with a~idin and on the other hand an a~idin complex wi.th the biotin--labled [)N~.
the a~din (or strepta~idin) concentra-tion is fixed, an i.ncrease in the concentrati.on of the a~idin--biotin-nucleic acid cornplex will cause a corr(:!spc)nding increase in the concent.ration of the free Biotin Cytochrolne-C species, which will cause a related increase in -the rate of -transfer of charge -from the enzyrne to the electrode surface.
~s an exarr1ple a gold electrodc! surface modified with 4,4-bipyridyl, or like rnaterial, was used. In this exemplary rnethod the biotin-labelled cytochrorne C acts as a rnediator to an enzyrne, in the presen-t exarnple, horsc!radish peroxidase.
Thc! enzyrne transfers charge bctween th~! mediator, ~ .
the oxiclised forrn of a mc!di.ator MoX (For ~!xample the ferriciniurn ion) to the reduced forrn Mr,!d (for exarnple ferrocence.). MrfCl is oxidised at the electrode G the current passed is rneasured and is proportional to the amount of subst.ratf! S presc!nt.
Bound to the enzyrne E by any sui-table rnethod ls a DN~
fragrrlf!nt D which may be c!ither deri~Jf.!d frorrl a naturally occuring DN~ sequence or rnay be synthetic. In the presencfe of an excf.!ss of the substratc! S a steady sta-te curren-t is obtained. ~ sarnple of DN~ which is to be assayed fc~r a parti.culclr -target sfequferlcf? complc!rrlentary to the sequence of the DN~ -fragrnr~ t D is con~ertecl to singl.e strands by any sui.table mf!thnd and then addf!d to the reaction rllixture.
Il: a targc!t sf!quf!ncE! cornplf!rrlf!ntary to the DN~ fragmf!nt D
is present in the rnixture it will b:ind to the fragrnent D and i.nhibit the enzyrrlatic rf!acti.on of the enzyrrlf! E
with the substrate S. Consequently the throughput of substrate S to product P will be reduced and the coupled reduction of Mox will be dirninished. The change in -the rate of reduction of M is reflf.!ctf!d in a rc!ducti.on o ;~
: of the current at the electrode G.
:Thr-! change i.n current is proportional to the arnount of the fragrnented DN~ D which is now bouncl -to target DN~
;..
and hence to thr* arnount of targr.!t DN~ presc!nt Examplc! 2; MEDI~TOR ~T~CHED TO DNQ PROBE
In exarnple B, the redox-ac tive rnediator M (in this exarrlplr.! a sllbsti. tut~!d fc!rroc~!nr!) is attachc!d dirc!c tly (as shown ln Figure 2) to the fragrnented DNh which is -to be used as a probe. The formation of mr!cliator-].inkecl DN~-probe cloes not impede eithrer -the arnperolne-tric respc)nsr! of -the medi.a-tor M nor l:.he b.inding interaction o-f the media-tor-DNh probe wi th the cornplernentar y target sequr!nce containr!d in -the DNQ which i.s being assayecl.
I he alnperornetric response cawsecl hy the addition of the medi.ator-DN~ probe to -the assay mi.xturr! is meas~rr-!d. If genetic rnaterial in a single- strandecl -forrn to which the probc! i s corrlplr!mr!ntary i s prr! s r!n t t~lr pl obr! bi nds to the cornplernentary sequence in the sample DN~. rhis great].y reducr-!s or~ completely inhibits the ampr!rc)rrle-tric response that is the complex of the rn(?diator-DN~ probe and t:he target DN~ is not arnperoTrletrically activr-!. The reduction in the initial arnperornetric response is in direct proportion to the amount of mr!diator-DN~
probe.itarget DN~ cornplex Forrned and- hence to the arnount of qenr!tic material containing a seqllr!nce cornplerrlentary to the known sequence of the rnediator-DN~ probe.
.... .
36;3~
_x_nple 3; MEDI~TOR-~Nr~_ INKER_~"Lr~ ED TO DN~_PROBE
In this r!xample (as shoon in Figurc! 3) the mr!diator-DNQ
probe also contains one or rnore linker groups L., (blotin n~ay for example be used).
rhe rnediator-linker-r~NQ probe is in this exernplary TTlr!thod trr!atr?d with the sarTlplr! suspc!c-tr!d of containing the single strancled genetic rnaterial which is the target for the DNQ probe ancl -the mr!diator--linkr!r-DNQ probe binds to any cornplelnentary sequence present, :
: ~n r;!'lect.rodr! G on whosr! sur-Face is present an elec-trochemical.ly acti~e rllaterial R, that recognises the li.nker group L, for exarrlplr! aui~in labellr!d witl ferrocene, is then irlllnersr!cl in -the reaction mixture.
The curren-t rrleas.lred on applying a potenti.al is re~uced on the binding of the electrochernically actiue rnaterial R to -the nnecliator-linker-DNQ probe /target-DN~ complex.
The recluction of the current is again ln propor-tion to the ~Tnount of th~! added singl~ strandr!d sc!qur!ncr! with a target sequence cornplernentary to the known sequence of the r~NQ-probe.
EXQMPLE _~ SYSTEM COMPRIJING ,BIOTIN C'~TOCHROME--C QND
P,,EROXIDQSE
In this particular ernbodirnent, the electrocle exh.ibits rc!~c!rsiblc! c!l~ctron transfc!r with biotin-labl.c!d cytochrorne-C. -rhe biotin-labled cytochrorne-C acts as a mediator compound, but can ~lsc) t~ind to a~idin as described in general terrns aboue.
The adcli.ti.on of biotin--labled nuc].eic acid ([)N~ or ~N~) to the rrlixture sets up a cornpeti-ti~e speci-fic binding rc!ac-ti.c)n bf!twc!c!n on thc! ~nc! hancl a Biotin Gytochrorr1c!--C
complex with a~idin and on the other hand an a~idin complex wi.th the biotin--labled [)N~.
the a~din (or strepta~idin) concentra-tion is fixed, an i.ncrease in the concentrati.on of the a~idin--biotin-nucleic acid cornplex will cause a corr(:!spc)nding increase in the concent.ration of the free Biotin Cytochrolne-C species, which will cause a related increase in -the rate of -transfer of charge -from the enzyrne to the electrode surface.
~s an exarr1ple a gold electrodc! surface modified with 4,4-bipyridyl, or like rnaterial, was used. In this exemplary rnethod the biotin-labelled cytochrorne C acts as a rnediator to an enzyrne, in the presen-t exarnple, horsc!radish peroxidase.
Thc! enzyrne transfers charge bctween th~! mediator, ~ .
3~
2~
(biotin cytochrorne C in this cas~!), and the substrate (H202), as shown by the cataly-tic current produced on addition of H202, as illustrated in Figures 4a and 4b Both the onzyrrle ancl the substrate are present i.n excess. rhe method operates by poislng the potential o-f the cathode at a suitable, negati.ve ~alue, gi~ing a resul-ting ca-taly-tic current which depc-!nds on the bi.otin-cytochrome C concentration.
Q~idin or strep-tauiclin is now aclded to the systern. Solne o-f the mediator complexes to th~ a~idin, and the ca-talytic currellt consequently clecreases The addition oF the bioti.ll~containing ~NQ or RNQ releases som~! of the biotin cytochrorne C since the forrner cornpetes -for the a~i.din pres~!nt ~s a resul.t the catalytic current increases in proportion of the added biotinyla-ted DN~, as illustrated in Figure 5.
Experirnental concditions Q con~entional three electrode systern, was empl-Jyed, wherein a 4,4-bipyridyldisulphide-coatecl gold electrode was us~!d as th~! working ~ ctrod~. Oth~r corrlpon~!nts of the assay mixture were 0.05M phosphate buffer, pH 6.2;
biotin-cytochrorne C (SIGMQ); 0~ 2 8m9; ~l2o2 2'~
300-80uM; horseradish peroxidase 210ug/ml; a~iclin 5-SOug; biotin polyuridyl tBRL) 1-lOul.
The cycli.c ~o1tamrnogram of biotin-cytochrorn~! C (scan between --200 and ~200rnU) does not change when the enzyrne horseradish peroxidaste is aclded to the cell, as can be seen -frorn Figure 4a.
le adcliti.c~n of substrate (H~C)2) results in a drarllatic change due to the ca-talytic curren-t, as shown in Fi.gure 4h.
Ihc~ !ctrocl~! is pois~!cd at a r~!ducti.on pot~!ntial as is shown generally in figure 5 the acldition of substrate as shown at 5a, produces the catalytic current which depends on the biotin cytochrolne C concelltration.
:
ln a second step, a~idin is added to the assay syst~rn;
the aclcli-tion of the sarne amoun-t of substrate results in reducecl catalytic current because some of th~
biotin-cytochrome C is colnplexed, as shown in Figure 5b In a third step as shown in figure 5c, Biotin--DNQ, RNQ
is adcled -to the systern addition oF the sarne arnount of H202 increasecd catalytic current thus showing a clependence on the probe b~ing present.
EX~MPLE 5 QC-rI~JQTED CQLF BIOTIN- LQBLLED CQLF-~'rHYMUS DNQ
This exarnple ernploys an electrode surf'ace which detc-~cts and rneasures in the presence of a mc!diator which in this caso is biotin cytochrorne C coualently a-ttached to r~N~ or RN~ or cy-tochrome C co~Jalently attachecl to ~N~ or RNQ an enzyrne -catalysed reac tion ~I'he cata'lytic current in the presence of ' excess ~!nzyme or substrate is a rneasure of the biotin--cytochrc)lne C- L)NQ(BC:[~NQ) or cytochrome DNQ(C[)NQ) present i.n the olectrochelllical cell.
Fxperim~!ntal. conditions for LJre~aration of acti~)a-tecl BC
.._. _. . .. _ ... _ . ___ _ . _ .._. .. .. . __ _ ...._ .. _. _ .. ._.. . _ _ _. _ .__.__ _ _ .. _ .
I lb~..lled cal f thy!nus DN~_ Q cytochrome C bridge was use(i to attachecl the bioti.n to r)NQ (Mannirlg et al) E3iotin--labell~cl cytochroml? C
(comrrlercially a~ailabl~! frorn SICM~) 5Irlg was acldecl 4-0 4 rn(3 of actilJated calk thyrnus DN~ (Pharrnacia PL
Biochemicals) in 0. lM triethanolarrline (TEQ) pH7 . 8 .
1.2rnl of 6% forrnaldehyde was added in the same buffer for cross-linking and th~ mixtllre was incubated at 3'1 for an hour . The forrnaldehycle was remo~Jed by extens iue dialysis .
.. . .
3~
Soclium chloride was added to the sarnple to g-l~e final concentration lM (to dissociate any cytochrome C which was not co~alently bound).
The soluti.on was fractionated on an FPLC gelfiltratirn colurnn, and using Sepharose 6B. The fract:ions were dc!tec-tc!d at ~54.nrrl. Thc! c!xtincti.on coc!f~icic!nt of ~Bcrrl2/rrlg for DNQ at 260nrrl, and 7.7 crn2/rr1g for biotin--cy-tochrorne C a-t 410nln were used -to calculate the nuc].eoti.de to cytochrome C ratio and was found approx.irrlately 20 1 in -the product.
The F3CL)NQ or C:DNQ acts :in the e].ectrocherrlical cell. as a rnediator, e.g., horseradish peroxidase; In the presence oF SllbS trate the catalytic current is a measure of the arnount of BCDNQ or CDN~ present or -the arnount of hybrid p ~ ! s (~! n t .
Thc! electrode i.s poised to a reduction poten-tial. l~he catalytic current indicates the presence of the biotin cytochrome C` DNQ (or RNQ) or cy tochrome C DN~ (or RN~) .
~ standard -three electrode system ernploying a 4-4 bipyridyldisulphide-coatecl gold working elc!ctrode was used in 0.05M phosphate bu-ffer at pH6.2. The rnedi~tor is Biotin-cytochrorne-DNQ (BCL.~N~) in this exarrlple, although it is en~isaged that any other electrocherrlical label attached through a cytochrorne bricdge which mr!diatr!s br!twr!en hors~!rac1ish pr!roxiclasr! and an ~ !ctrodFJ
could be used.
If cytochrornr! C i.s usr!c1, its concentra-ti.on shoul-,1 be not rnore than 300uM because of the c1anger of b1~achin(3 the cytochromr.!--~, at higher concentrations.
EX~MP1-E 6, N:rCK TRQNS1~"1-:rON OF B.IO'rlN LQBLLED 'PH~GE
....... _._.. _._._.. ___.. __.. ___.. _ __.. __._______._._._.. _.. ____.. __.. ___.. _.. ____,.. _._.. ,.__.. _.____.. __.. ___.. _____.. __.. __._.
. _.. _._ L~MD~ DN~
_ .... _...... _. _ .
In tllis r!xr~!rrlpl.clry rrlr!thod, ~Isr! is 1r1ad r-! C1 f an F! ¦ r! ctr~c~dr!
surface which can detect or rneasure, in the presence of a mecliator, an enzyn1r! comp1r!x-cat:a1ysrSc1 reaction. In -this exarnple the enzyrne cor1lplex consists of biotin peroxi.c1asF!-a~idin in a proporti.on such that i.t can bF.!
bind aclditional biotin or a biotin containi1lg r1la-teria1 such as hi.otin-lab-!llr.!d DN~ c~r ~N~.
~ steady-state curren-t is obtained using the biotin pr!roxi.dasr!-a~idin cornplr!x in thr! prPsr!ncr! oF ~122 ancl a rnediator such as cytochrorne C, a ferrocene corr1pound or, in thr! r!xarrlplF.!s shown ~lr!rr!in [Ru(N~13)5 pyJ2'~ ~h.! r!1-!ctroc1r! is pois-!d at a nr!gati~r!
potential. Thr! substrate, H~02 is present in high excess, The current clecreases when biotin label1ed DN~
o~ RN~ is addecl to thc! cc!11, The biotin DN~ may be is fi3~
either in solution or is attached to a rn~lnbrane, Qt a limiting concentration of thr! biotin peroxidase--auidin colnplex and excess rnecdiator and substrate, if thP r!lr!ctrodr! is poishd at, a suitahlh potential, (in this particular instance negatiue with rr!spr!ct to a sa-turathd c~llorllr!]. c!lr!çtrodrl) a str!ady sta-th çurrent is obtained whlch ls propor-tional to thr..! enzyrne concc-!ntra-tion .
From thi.s poir~t thh r!xpr!rirrlr!nt prc!sr!ntr!d hc!re by Wcly o-f exall1plr! was perforlnecl according to two cliffelAent r!xpr~r-l.rrlr!nt~A]. protocol.s, that i.5 r!i thr!lA b~ thr! ~clclition o l~;
a) E3iotin DNQ or E3iotin RN~ added in solution , b) :rnllnobilisr!d, biotin DN~ or hiotin RNQ is acdded In hoth instances the nuclr!ic aci.d reacts with the enzyrne complex, and giues a current increase which is proportional to, and acts as a mc!asure of, the adcled E3iotin DNh or RN~.
Ex~erimr!ntal conditions for thh Nick translation of __. _.. --.~_.__ _.. _ __ _ .. __ .. _ ._ .. .... __.. _. _._.___ _ __.. ._._ __ h,iotin-labellr~.d Ph~a~e_Landa DNh_ ' Sonicated phage Larnda DN~ was nick translated in the presence of biotin~ dUTp accorcling to the 5Rl technical inforrna-tion protocol using their nick trans].ation reagent system the reagents errIployecI being as follows;
1) lOul solution of the rnixture of O.~mM dnTp~ dCTP
dGl'P in 500rnM trls pH7.8 50InM MgCl2 100InM
2-rnercaptoethanol and lOOIng/Inl nuclease--free 5S~
2) 4ug of L..amda phag~! DN~ in 0.1mM E[)T~ 10mM tris pH:7.5 and 100rrIM socIluIn chloride.
3) ].Oul. of t~io-tin~ IUTP in 0.4rTIM in IOOTTIM tr~is p~I 7.5.
"I'he ~olurne was rnade up to ~ou1 and the reaction rnixtlIre was carefu].ly rmixed.
2~
(biotin cytochrorne C in this cas~!), and the substrate (H202), as shown by the cataly-tic current produced on addition of H202, as illustrated in Figures 4a and 4b Both the onzyrrle ancl the substrate are present i.n excess. rhe method operates by poislng the potential o-f the cathode at a suitable, negati.ve ~alue, gi~ing a resul-ting ca-taly-tic current which depc-!nds on the bi.otin-cytochrome C concentration.
Q~idin or strep-tauiclin is now aclded to the systern. Solne o-f the mediator complexes to th~ a~idin, and the ca-talytic currellt consequently clecreases The addition oF the bioti.ll~containing ~NQ or RNQ releases som~! of the biotin cytochrorne C since the forrner cornpetes -for the a~i.din pres~!nt ~s a resul.t the catalytic current increases in proportion of the added biotinyla-ted DN~, as illustrated in Figure 5.
Experirnental concditions Q con~entional three electrode systern, was empl-Jyed, wherein a 4,4-bipyridyldisulphide-coatecl gold electrode was us~!d as th~! working ~ ctrod~. Oth~r corrlpon~!nts of the assay mixture were 0.05M phosphate buffer, pH 6.2;
biotin-cytochrorne C (SIGMQ); 0~ 2 8m9; ~l2o2 2'~
300-80uM; horseradish peroxidase 210ug/ml; a~iclin 5-SOug; biotin polyuridyl tBRL) 1-lOul.
The cycli.c ~o1tamrnogram of biotin-cytochrorn~! C (scan between --200 and ~200rnU) does not change when the enzyrne horseradish peroxidaste is aclded to the cell, as can be seen -frorn Figure 4a.
le adcliti.c~n of substrate (H~C)2) results in a drarllatic change due to the ca-talytic curren-t, as shown in Fi.gure 4h.
Ihc~ !ctrocl~! is pois~!cd at a r~!ducti.on pot~!ntial as is shown generally in figure 5 the acldition of substrate as shown at 5a, produces the catalytic current which depends on the biotin cytochrolne C concelltration.
:
ln a second step, a~idin is added to the assay syst~rn;
the aclcli-tion of the sarne amoun-t of substrate results in reducecl catalytic current because some of th~
biotin-cytochrome C is colnplexed, as shown in Figure 5b In a third step as shown in figure 5c, Biotin--DNQ, RNQ
is adcled -to the systern addition oF the sarne arnount of H202 increasecd catalytic current thus showing a clependence on the probe b~ing present.
EX~MPLE 5 QC-rI~JQTED CQLF BIOTIN- LQBLLED CQLF-~'rHYMUS DNQ
This exarnple ernploys an electrode surf'ace which detc-~cts and rneasures in the presence of a mc!diator which in this caso is biotin cytochrorne C coualently a-ttached to r~N~ or RN~ or cy-tochrome C co~Jalently attachecl to ~N~ or RNQ an enzyrne -catalysed reac tion ~I'he cata'lytic current in the presence of ' excess ~!nzyme or substrate is a rneasure of the biotin--cytochrc)lne C- L)NQ(BC:[~NQ) or cytochrome DNQ(C[)NQ) present i.n the olectrochelllical cell.
Fxperim~!ntal. conditions for LJre~aration of acti~)a-tecl BC
.._. _. . .. _ ... _ . ___ _ . _ .._. .. .. . __ _ ...._ .. _. _ .. ._.. . _ _ _. _ .__.__ _ _ .. _ .
I lb~..lled cal f thy!nus DN~_ Q cytochrome C bridge was use(i to attachecl the bioti.n to r)NQ (Mannirlg et al) E3iotin--labell~cl cytochroml? C
(comrrlercially a~ailabl~! frorn SICM~) 5Irlg was acldecl 4-0 4 rn(3 of actilJated calk thyrnus DN~ (Pharrnacia PL
Biochemicals) in 0. lM triethanolarrline (TEQ) pH7 . 8 .
1.2rnl of 6% forrnaldehyde was added in the same buffer for cross-linking and th~ mixtllre was incubated at 3'1 for an hour . The forrnaldehycle was remo~Jed by extens iue dialysis .
.. . .
3~
Soclium chloride was added to the sarnple to g-l~e final concentration lM (to dissociate any cytochrome C which was not co~alently bound).
The soluti.on was fractionated on an FPLC gelfiltratirn colurnn, and using Sepharose 6B. The fract:ions were dc!tec-tc!d at ~54.nrrl. Thc! c!xtincti.on coc!f~icic!nt of ~Bcrrl2/rrlg for DNQ at 260nrrl, and 7.7 crn2/rr1g for biotin--cy-tochrorne C a-t 410nln were used -to calculate the nuc].eoti.de to cytochrome C ratio and was found approx.irrlately 20 1 in -the product.
The F3CL)NQ or C:DNQ acts :in the e].ectrocherrlical cell. as a rnediator, e.g., horseradish peroxidase; In the presence oF SllbS trate the catalytic current is a measure of the arnount of BCDNQ or CDN~ present or -the arnount of hybrid p ~ ! s (~! n t .
Thc! electrode i.s poised to a reduction poten-tial. l~he catalytic current indicates the presence of the biotin cytochrome C` DNQ (or RNQ) or cy tochrome C DN~ (or RN~) .
~ standard -three electrode system ernploying a 4-4 bipyridyldisulphide-coatecl gold working elc!ctrode was used in 0.05M phosphate bu-ffer at pH6.2. The rnedi~tor is Biotin-cytochrorne-DNQ (BCL.~N~) in this exarrlple, although it is en~isaged that any other electrocherrlical label attached through a cytochrorne bricdge which mr!diatr!s br!twr!en hors~!rac1ish pr!roxiclasr! and an ~ !ctrodFJ
could be used.
If cytochrornr! C i.s usr!c1, its concentra-ti.on shoul-,1 be not rnore than 300uM because of the c1anger of b1~achin(3 the cytochromr.!--~, at higher concentrations.
EX~MP1-E 6, N:rCK TRQNS1~"1-:rON OF B.IO'rlN LQBLLED 'PH~GE
....... _._.. _._._.. ___.. __.. ___.. _ __.. __._______._._._.. _.. ____.. __.. ___.. _.. ____,.. _._.. ,.__.. _.____.. __.. ___.. _____.. __.. __._.
. _.. _._ L~MD~ DN~
_ .... _...... _. _ .
In tllis r!xr~!rrlpl.clry rrlr!thod, ~Isr! is 1r1ad r-! C1 f an F! ¦ r! ctr~c~dr!
surface which can detect or rneasure, in the presence of a mecliator, an enzyn1r! comp1r!x-cat:a1ysrSc1 reaction. In -this exarnple the enzyrne cor1lplex consists of biotin peroxi.c1asF!-a~idin in a proporti.on such that i.t can bF.!
bind aclditional biotin or a biotin containi1lg r1la-teria1 such as hi.otin-lab-!llr.!d DN~ c~r ~N~.
~ steady-state curren-t is obtained using the biotin pr!roxi.dasr!-a~idin cornplr!x in thr! prPsr!ncr! oF ~122 ancl a rnediator such as cytochrorne C, a ferrocene corr1pound or, in thr! r!xarrlplF.!s shown ~lr!rr!in [Ru(N~13)5 pyJ2'~ ~h.! r!1-!ctroc1r! is pois-!d at a nr!gati~r!
potential. Thr! substrate, H~02 is present in high excess, The current clecreases when biotin label1ed DN~
o~ RN~ is addecl to thc! cc!11, The biotin DN~ may be is fi3~
either in solution or is attached to a rn~lnbrane, Qt a limiting concentration of thr! biotin peroxidase--auidin colnplex and excess rnecdiator and substrate, if thP r!lr!ctrodr! is poishd at, a suitahlh potential, (in this particular instance negatiue with rr!spr!ct to a sa-turathd c~llorllr!]. c!lr!çtrodrl) a str!ady sta-th çurrent is obtained whlch ls propor-tional to thr..! enzyrne concc-!ntra-tion .
From thi.s poir~t thh r!xpr!rirrlr!nt prc!sr!ntr!d hc!re by Wcly o-f exall1plr! was perforlnecl according to two cliffelAent r!xpr~r-l.rrlr!nt~A]. protocol.s, that i.5 r!i thr!lA b~ thr! ~clclition o l~;
a) E3iotin DNQ or E3iotin RN~ added in solution , b) :rnllnobilisr!d, biotin DN~ or hiotin RNQ is acdded In hoth instances the nuclr!ic aci.d reacts with the enzyrne complex, and giues a current increase which is proportional to, and acts as a mc!asure of, the adcled E3iotin DNh or RN~.
Ex~erimr!ntal conditions for thh Nick translation of __. _.. --.~_.__ _.. _ __ _ .. __ .. _ ._ .. .... __.. _. _._.___ _ __.. ._._ __ h,iotin-labellr~.d Ph~a~e_Landa DNh_ ' Sonicated phage Larnda DN~ was nick translated in the presence of biotin~ dUTp accorcling to the 5Rl technical inforrna-tion protocol using their nick trans].ation reagent system the reagents errIployecI being as follows;
1) lOul solution of the rnixture of O.~mM dnTp~ dCTP
dGl'P in 500rnM trls pH7.8 50InM MgCl2 100InM
2-rnercaptoethanol and lOOIng/Inl nuclease--free 5S~
2) 4ug of L..amda phag~! DN~ in 0.1mM E[)T~ 10mM tris pH:7.5 and 100rrIM socIluIn chloride.
3) ].Oul. of t~io-tin~ IUTP in 0.4rTIM in IOOTTIM tr~is p~I 7.5.
"I'he ~olurne was rnade up to ~ou1 and the reaction rnixtlIre was carefu].ly rmixed.
4) 10ul. DN~ polymerase was added to the mixture 0.4 units per ul 40 pg per rnl r~N~ase I in 50rnM tris p~l7.5 5TrlM magnesiuTrl acetate, lmM ~-mercaptoethanol, O.lrnM
phenylmethyl-sulfonylfluoride 50% glycerol and 100ug per ml nuclease frr-!e BS~.
The mixture was incubated at 15C for 90 miIlutes. 'rhe reaction was stopped by addiing lOu]. 300mM EDT'~. T'he purification of thc biotin-labelled DN~ was carried out ; 3 ~
according to the "Spin colurnn Procedure" -for Maniatis et al [Mantiati, F'ritsor and Sambrook, Molecular Cloning a laboratory rnanual, 1~2, Cold Spring Harbor Labs 466-467~.
~ 1-rnl disposable syringe was used, whi.ch was plugged with a srnall arnount oF glass woo].. ~ 0,9lnl bed uolurne of Sc!phadex oas ecluili.bratecl wi.th 0.15M sodium chlori.de, ancl O.O].SM sodillln ci-trate at p~7,0 was packed in the syringe, and was spun on a bench centrifucJc!, se~eral times until the packc-!d uolurne was O.9lnl. O.lrnl of b~lfFer was therl added to the syringe and spun at the salne speed and for the sarne time.
F'ina].'l.y, th~! nick translati.on mixture was addcd in O.lml ancl re-cen-tr.ifuged at exactly the same spec-!d and -tirne and thc! IOOul of ~!ffFluc!nt was collc!ctc!d i.n a dc!~-cappc!cd Eppendorf tube, which contained the biotin-labelled phage Lamcla DN~. The biotin-labell.ed phage L.amda DN~
~RL, probed, nick translated, as aboue, and biotin cytochrome C--labelled acticuated ca].fF thyrnus ~N~ were attached to the nitrocellulose rnernbrane by heating at 80~C for 2 hours in a ~acuurn o~en.
EX~MPLE 7; E.LECTROCHEMIC~L_ DETECT'ION OF _ IOT:CN-L~BLI.ED
L~MD~ DN~
i3~
This exp~rirnent was perforrned in two ways, with the r~Nh in solution and the DN~ bound to a nitrocellulose rnernbrane.
In bot.h cases the assay systerTI cornprised a gold electrode in a 0.7ml uolulne cell T'he reagents elTlployed for this exarnple were;
0,05M phosphate buffer pH 6.2 Mediator a) 0.15 mM ferroc~!ne monocarboxylic acid b) 0.16rnM [Ru(NH3)5]Pyr2~
Bio-tin-peroxidase-auidin enzyme cornplex concen-tra-tion 60nM-O.~nM as stated.
8.81nM 1l202 as substrate.
'T'he Forrrlation of enzyme complex was aCCornpliShF!d .lS
follows: uecstatin ~BC (seralab) was ~sed und~!r their recc)rrlrnended conditions; 50ul of bio-tin-pc!roxidase 5mg/rrll was incuhated with 50ul of Quidin lrng/rnl in lml assay buFfer containing 0.1% 'rween 20 for 30 minutes. -rhe further dilutions were rnade in the assay buffer cc)ntaining 0.1% Tween 20.
EX~MPLE 7a- USE OF FERROCENE MONOC~RBOXYI:[C ~CID
When ferrocene monocarboxylic acid was used as a rnediator, the catalytic current is linear under the ~3 ~' ?~ O L` ''~
~l~ ~, ~ ~Y~
condition frorn 60nM to 6nM enzyrne concentration gi~ing a catalytic current in the rage of 0.~ - 0.7u~.
EX~MPLE 7b-- USE OF RUTIlENIUM COMPLEX ~S ~ MEDI~TOR
If [PyRu(NH3)5l i.s usecl as mecli.ator, the catalyti.c curr~nt i5 approxirrlately tenfold lclrger than in the pre~ious case, therefore it was the choice in -the thre~! s~lbsicluary ~!xp~!rirrl~!nts d~!scrib~!d by way of ~!xarrlpl~
below.
EX~MPL.E 7c-E`NZYME (:~NCFNrR~IC)N 6 0nMO_~R
. In this exalnple the enzyrne concentration was 6nM. In th~! pr~!s~!nc~! of H202 a st~!ady stat~! catalyti.c cl.lrrent in observcd, as shown in Figure ~a.
When biotin labelled phage Larncla DN~ 200ng is added to th~ !ctrochcrrlical ccl]. and thc stcady sta-t~! catalytic current decreased. This is illustrated in Figure 8b.
Further injection of H202 s-till results in a catalytic currcnt. The result shows that the biotin-DN~
cornplexes sorne of the biotin a~idin enzyrne cornplex, but still ther~ is a high ~Xc~!ss of th~! ~!nzyrrl~! cornplcx present which did not bind to the biotin DN~.
~ ;.
~'s',23~3~
EX~MPLE 7d;-ENZYME CONCENTR~TION 0.6 nMOL~R
This exampl~! follows th~ lin~!s of the pre~ious exarnple the only change being in the enzyrne cornplex conc~!ntration, whi.ch is 0.6nM i.n chis cas~w rhe catalytic current, as shown in Fig. 7b, characteri.stic of this enzyrrle complex concentration drastically changes, as shown in Figure 7c, after addition of B-lotill DN~ (ZOOug). With further l-i202 injection the current rernains at a decreased le~el as illustrated i.n fi.gure 7D.
.
EX~MPlE 7e;-DN~ IMMOBlLlZrD ON_MFME~R~NE.
catal.ytic current is oh-tai.necl with H202 substantially as descibed abo~Je. rlle biot:in-labellecl DN~
imrrlobili.s~!cl on a nitrocell.ulos~ membrane, was acldE!d to the cell ln the range of 0.5.ug -- 0.2 ug of DN~. ~fter mi.nut~s incubation with -th~! biotin DN~ merrlbrane a f`urther H~02 injection did not induce any catalytic curr~!nt, as shown by figure 7~!, thus indicating that the enzyrne cornplex bincls to the biotin DN~ present on the mernhran~!, and charg~! transf~!r i.s th~!r~!for~! inhibit~!d.
phenylmethyl-sulfonylfluoride 50% glycerol and 100ug per ml nuclease frr-!e BS~.
The mixture was incubated at 15C for 90 miIlutes. 'rhe reaction was stopped by addiing lOu]. 300mM EDT'~. T'he purification of thc biotin-labelled DN~ was carried out ; 3 ~
according to the "Spin colurnn Procedure" -for Maniatis et al [Mantiati, F'ritsor and Sambrook, Molecular Cloning a laboratory rnanual, 1~2, Cold Spring Harbor Labs 466-467~.
~ 1-rnl disposable syringe was used, whi.ch was plugged with a srnall arnount oF glass woo].. ~ 0,9lnl bed uolurne of Sc!phadex oas ecluili.bratecl wi.th 0.15M sodium chlori.de, ancl O.O].SM sodillln ci-trate at p~7,0 was packed in the syringe, and was spun on a bench centrifucJc!, se~eral times until the packc-!d uolurne was O.9lnl. O.lrnl of b~lfFer was therl added to the syringe and spun at the salne speed and for the sarne time.
F'ina].'l.y, th~! nick translati.on mixture was addcd in O.lml ancl re-cen-tr.ifuged at exactly the same spec-!d and -tirne and thc! IOOul of ~!ffFluc!nt was collc!ctc!d i.n a dc!~-cappc!cd Eppendorf tube, which contained the biotin-labelled phage Lamcla DN~. The biotin-labell.ed phage L.amda DN~
~RL, probed, nick translated, as aboue, and biotin cytochrome C--labelled acticuated ca].fF thyrnus ~N~ were attached to the nitrocellulose rnernbrane by heating at 80~C for 2 hours in a ~acuurn o~en.
EX~MPLE 7; E.LECTROCHEMIC~L_ DETECT'ION OF _ IOT:CN-L~BLI.ED
L~MD~ DN~
i3~
This exp~rirnent was perforrned in two ways, with the r~Nh in solution and the DN~ bound to a nitrocellulose rnernbrane.
In bot.h cases the assay systerTI cornprised a gold electrode in a 0.7ml uolulne cell T'he reagents elTlployed for this exarnple were;
0,05M phosphate buffer pH 6.2 Mediator a) 0.15 mM ferroc~!ne monocarboxylic acid b) 0.16rnM [Ru(NH3)5]Pyr2~
Bio-tin-peroxidase-auidin enzyme cornplex concen-tra-tion 60nM-O.~nM as stated.
8.81nM 1l202 as substrate.
'T'he Forrrlation of enzyme complex was aCCornpliShF!d .lS
follows: uecstatin ~BC (seralab) was ~sed und~!r their recc)rrlrnended conditions; 50ul of bio-tin-pc!roxidase 5mg/rrll was incuhated with 50ul of Quidin lrng/rnl in lml assay buFfer containing 0.1% 'rween 20 for 30 minutes. -rhe further dilutions were rnade in the assay buffer cc)ntaining 0.1% Tween 20.
EX~MPLE 7a- USE OF FERROCENE MONOC~RBOXYI:[C ~CID
When ferrocene monocarboxylic acid was used as a rnediator, the catalytic current is linear under the ~3 ~' ?~ O L` ''~
~l~ ~, ~ ~Y~
condition frorn 60nM to 6nM enzyrne concentration gi~ing a catalytic current in the rage of 0.~ - 0.7u~.
EX~MPLE 7b-- USE OF RUTIlENIUM COMPLEX ~S ~ MEDI~TOR
If [PyRu(NH3)5l i.s usecl as mecli.ator, the catalyti.c curr~nt i5 approxirrlately tenfold lclrger than in the pre~ious case, therefore it was the choice in -the thre~! s~lbsicluary ~!xp~!rirrl~!nts d~!scrib~!d by way of ~!xarrlpl~
below.
EX~MPL.E 7c-E`NZYME (:~NCFNrR~IC)N 6 0nMO_~R
. In this exalnple the enzyrne concentration was 6nM. In th~! pr~!s~!nc~! of H202 a st~!ady stat~! catalyti.c cl.lrrent in observcd, as shown in Figure ~a.
When biotin labelled phage Larncla DN~ 200ng is added to th~ !ctrochcrrlical ccl]. and thc stcady sta-t~! catalytic current decreased. This is illustrated in Figure 8b.
Further injection of H202 s-till results in a catalytic currcnt. The result shows that the biotin-DN~
cornplexes sorne of the biotin a~idin enzyrne cornplex, but still ther~ is a high ~Xc~!ss of th~! ~!nzyrrl~! cornplcx present which did not bind to the biotin DN~.
~ ;.
~'s',23~3~
EX~MPLE 7d;-ENZYME CONCENTR~TION 0.6 nMOL~R
This exampl~! follows th~ lin~!s of the pre~ious exarnple the only change being in the enzyrne cornplex conc~!ntration, whi.ch is 0.6nM i.n chis cas~w rhe catalytic current, as shown in Fig. 7b, characteri.stic of this enzyrrle complex concentration drastically changes, as shown in Figure 7c, after addition of B-lotill DN~ (ZOOug). With further l-i202 injection the current rernains at a decreased le~el as illustrated i.n fi.gure 7D.
.
EX~MPlE 7e;-DN~ IMMOBlLlZrD ON_MFME~R~NE.
catal.ytic current is oh-tai.necl with H202 substantially as descibed abo~Je. rlle biot:in-labellecl DN~
imrrlobili.s~!cl on a nitrocell.ulos~ membrane, was acldE!d to the cell ln the range of 0.5.ug -- 0.2 ug of DN~. ~fter mi.nut~s incubation with -th~! biotin DN~ merrlbrane a f`urther H~02 injection did not induce any catalytic curr~!nt, as shown by figure 7~!, thus indicating that the enzyrne cornplex bincls to the biotin DN~ present on the mernhran~!, and charg~! transf~!r i.s th~!r~!for~! inhibit~!d.
Claims (28)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An assay for nucleic acid which comprises the steps of; (a) providing a probe material comprising; (i) a sequence of nucleic acids complementary to a given target sequence and, (ii) a first ligand chemically linked thereto and capable of a specific binding reaction with an antiligand; (b) contacting the said probe material with an assay system comprising; (i) a suitable mediator, enzyme, substrate system capable of transferring charge to an electrode surface when the enzyme is catalyticaly active, and; (ii) a second ligand chemically linked to one of said mediator, enzyme or substrate, wherein the second ligand is capable of a competitive binding reaction with the antiligand, and; (iii) the said antiligand, whereby the said first ligand competes with the said second ligand in a specific binding reaction with the antiligand, and;
(c) contacting the above system with a solution suspected of containing the said target sequence whereby the binding of any of the said target sequence present to the probe affects the availability of the first ligand and therefore alters the rate of charge transfer to the electrode.
(c) contacting the above system with a solution suspected of containing the said target sequence whereby the binding of any of the said target sequence present to the probe affects the availability of the first ligand and therefore alters the rate of charge transfer to the electrode.
2) The assay of claim 1, wherein the nucleic acid is deoxyribonucleic acid.
3) The assay of claim 1, wherein the nucleic acid is ribonucleic acid.
4) The assay of claim 1, wherein the specific binding reaction between either the first or the second ligand and the antiligand is characteristic of the specific binging reaction between a pair of reagents or derivatives thereof selected from the group of pairs comprising; biotin and avidin, antigen and antibody, hormone and receptor, lectin and carbohydrate, cofactor and enzyme, nucleic acid and complementary nucleic acid.
5) The assay of claim 4, wherein both the first ligand and the second ligand are biotin or derivatives thereof and the antiligand is avidin or a derivative thereof.
6) The assay of claim 1, wherein the mediator is an organometallic compound.
7) The assay of claim 6, wherein the mediator comprises at least one organic ring, which is characterised by at least two double bonds that are conjugated and a metal atom in electron-sharing contact with each of the rings.
8) The assay of claim 7, wherein the metal is a transition metal.
9) The assay of claim 8, wherein the metal is selected from the group comprising iron, chromium and ruthenium.
10) The assay of claim 9, wherein the mediator is a ferrocene.
11) The as say of claim 1, wherein the mediator is selected from the group comprising viologens, poly-viologens, phenazines, ferricyanide and derivatives therof, metalloporphyrins and one-dimensional conductors.
12) The assay of claim 1, wherein the mediator is a carboborane.
13) The assay of claim 1, wherein the mediator is a ruthenium compound.
14. The assay of claim 10, wherein the mediator is selected from the group consisting of ferrocene; chloroferrocene;
methyl-trimethylaminoferrocene; 1,1-dimethylferrocene; 1,1-dicar-boxyferrocene; carboxyferrocene; vinylferrocene, trimethylamino-ferrocene; 1,1 dimethylferrocene; polyvinyl-ferrocene; ferrocene monocarboxylic acid; hydroxyethylferrocene; acetoferrocene; and 1,1-bis-hydroxymethyl ferrocene.
methyl-trimethylaminoferrocene; 1,1-dimethylferrocene; 1,1-dicar-boxyferrocene; carboxyferrocene; vinylferrocene, trimethylamino-ferrocene; 1,1 dimethylferrocene; polyvinyl-ferrocene; ferrocene monocarboxylic acid; hydroxyethylferrocene; acetoferrocene; and 1,1-bis-hydroxymethyl ferrocene.
15. The assay of claim 11, wherein the mediator is a cytochrome.
16. The assay of claim 15, wherein the mediator is cytochrome-C.
17. The assay of claim 11, wherein the mediator is TONQ
18. The assay of claim 1 wherein the mediator is con-fined to an electrically conductive surface.
19. The assay of claim 1 wherein the enzyme is a non-oxygen specific flavo-protein.
20. The assay of claim 19, wherein said flavo-protein is selected from the group consisting of methanol oxidase, pyru-vate oxidase (EC 1.2.3.3.), xanthine oxidase (EC 1.2.3.2.), sar-cosine oxidase (EC 1.5.3.1.), lipoamide dehydrogenase (EC
1.6.3.4.), glutathione reductase (EC 1.6.4.2.) carbonmonoxide oxido-reductase, glucose oxidase, glycollate oxidase (EC
1.1.3.1.) L-amino acid oxidase (EC 1.4.3.2.) and lactose oxidase.
1.6.3.4.), glutathione reductase (EC 1.6.4.2.) carbonmonoxide oxido-reductase, glucose oxidase, glycollate oxidase (EC
1.1.3.1.) L-amino acid oxidase (EC 1.4.3.2.) and lactose oxidase.
21. The assay of claim 1 wherein the enzyme is a quino-protein.
22. The assay of claim 21, wherein said quino-protein is selected from the group consisting of glucose dehydrogenase alcohol dehydrogenase and methanol dehydrogenase.
23. The assay of claim 1 wherein the enzyme is confined to an electrically conductive surface.
24. The assay of claim 1 wherein the enzyme is a haem-containing enzyme.
25. The assay of claim 24, wherein the haem-containing enzyme is chosen from the group consisting of lactate dehydroge-nase yeast cytochrome-C peroxidase and horseradish peroxidase.
26. The assay of claim 1 wherein the enzyme is a cupro-protein.
27. The assay of claim 26, wherein the cuproprotein is galactose oxidase.
28. The assay of claim 1 wherein the electrode is made of a material chosen from the group comprising; gold, platinum, silver, carbon or a one-dimensional conductor.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8333651 | 1983-12-16 | ||
| GB838333650A GB8333650D0 (en) | 1983-12-16 | 1983-12-16 | Homogeneous enzyme immundassay techniques |
| GB8333650 | 1983-12-16 | ||
| GB838333651A GB8333651D0 (en) | 1983-12-16 | 1983-12-16 | Nucleic acid sequence probes in amperometric biosensors |
| GB848401399A GB8401399D0 (en) | 1984-01-19 | 1984-01-19 | Electrochemical immunoassay |
| GB8401399 | 1984-01-19 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1223639A true CA1223639A (en) | 1987-06-30 |
Family
ID=27262238
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000470321A Expired CA1223639A (en) | 1983-12-16 | 1984-12-17 | Assay for nucleic acids |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US4840893A (en) |
| EP (1) | EP0149339B1 (en) |
| AU (1) | AU583258B2 (en) |
| CA (1) | CA1223639A (en) |
| DE (1) | DE3479522D1 (en) |
| WO (1) | WO1985002627A1 (en) |
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- 1984-12-14 US US06/769,629 patent/US4840893A/en not_active Expired - Lifetime
- 1984-12-14 EP EP84308773A patent/EP0149339B1/en not_active Expired
- 1984-12-14 AU AU38329/85A patent/AU583258B2/en not_active Ceased
- 1984-12-17 CA CA000470321A patent/CA1223639A/en not_active Expired
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| AU583258B2 (en) | 1989-04-27 |
| US4840893A (en) | 1989-06-20 |
| EP0149339A2 (en) | 1985-07-24 |
| WO1985002627A1 (en) | 1985-06-20 |
| AU3832985A (en) | 1985-06-26 |
| EP0149339A3 (en) | 1985-08-21 |
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