CA2198489A1 - Biosensor for and method of electrogenerated chemiluminescent detection of nucleic acid adsorbed to a solid surface - Google Patents
Biosensor for and method of electrogenerated chemiluminescent detection of nucleic acid adsorbed to a solid surfaceInfo
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- CA2198489A1 CA2198489A1 CA002198489A CA2198489A CA2198489A1 CA 2198489 A1 CA2198489 A1 CA 2198489A1 CA 002198489 A CA002198489 A CA 002198489A CA 2198489 A CA2198489 A CA 2198489A CA 2198489 A1 CA2198489 A1 CA 2198489A1
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- 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
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- 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
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
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- 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
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- 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
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- 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
- C12Q1/6869—Methods for sequencing
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Abstract
Single-strand DNA was immobilized on an electrode covered with an aluminum alkanebisphosphonate film by immersing it in an ss-DNA solution. The immobilized ss-DNA labeled with Ru(bpy)32+ was detected by monitoring the electrogenerated chemiluminescence (ECL) produced upon oxidation in a solution containing tri-n-propylamine. After immobilization of unlabeled ss-DNA, a complementary labeled strand DNA was hybridized to produce ds-DNA on the surface. The extent of DNA hybridization was determined by ECL of the labeled Ru(bpy)32+. Surface immobilized ds-DNA could also be detected by observing the ECL of intercalated Ru(phen)32+. Transmission electron microscopy (TEM) was employed to image the film and the immobilized DNA.
Description
~ W096/06946 2 t q 8 4 8 9 Title of the Invention ~In~PN8oR FOR AND ~ETHOD OF T~'T,FI ,.~O~.~:N ~ TT~n r~T.~TT" ~N~ L ~ cLloN OF NUCLEIC ACID
~nSrRRTn TO A SOLID SURFACE
Field Of Invention The present invention relates to the diagnostic field, and pcppc;Ally nucleic acid diagnostics. In particular, the present invention relates to a probe or sensor having a film containing metal centers, its preparation where a single _LLGnd or double-strand nucleic acid sequence i5 i h;l;7P~ thereon on, and its use in the subsequent detection of the nucleic acid by lAhPl 1 ing with lnm;nPccPnt metal chelates.
In addition to using the subject hi oSPnC~r for nucleic acid diagnostics based on a surface designed modified electrical sensor, i.e., chips or electrode, ; -hili7ation and hybridization of nucleic acid such as DNA on a self-assembled thin film via suriace reaction is also useful in studying molecular recognition of DNA.
Back~round Of Invention Nucleic acid diagnostics has become an important area in molecular biology and biotechnology studies, with applications to the determinations of disease and food contaminating organisms and in forensic and enviLl LG1 investigations. The development of new DNA hiosPnc~rs has led to the application of several detection techniques such as W096/06946 2 1 9 8 4 8 9 PCT~S9~/10630 ~
optical methods (e.g., lnminpccpnre~ Pll;t y and pseudo-Brewster angle rPflectl y), piP7oPlPotric devices (e.g., SAW, QCM), and electrorhpm;rAl technigues (e.g., CV and SWV).
of particular interest are labels which can be made to lllm;npcre through phorrrhPmicnl~ rhPm;c~l, and electrorhDmi~nl reaction schemes. In particular, ele~LLu~l~ ilnm;nPcrPn~
methods of ~Dtprmining the presence of l~hPllD~ materials are preferred over other methods for many reasons. They are highly diagnostic of the presence of a particular label, sensitive, nonhazardous, inpyppnc;ve and can be used in a wide variety of applications. Suitable labels comprise ele~LLu~ llminPcrPnt , inrlll~;nrJ organic and metal chelates.
For example, electrorhpmil~lm;npcrpnt ruthenium- and osmiu~ . I~ininrJ labels have been used in methods for tPrting and guantifying analytes of interest in liguid medin (u~s~ Patent Nos. 5,310,687; 5,238,808; and 5,221,605). In addition, the application of ele~Lu~nerated chemi-lnminPccPnre ~ECL) mea~uL to the detection of solution phase DNA intercalated with ruthenium-containing labels has been described (Carter, M.T. et al. (1990) Bioconiu~ate Chem 2:257-263). However, reaction schemes that are viable in the solution phase are often not applicable in the solid phase.
More importantly, the detection of solution phase analytes such a5 DNA has several drawbacks relative to detection of analytes absorbed to solid surfaces. The advantages for detecting DNA via solid phase techni~ues as opposed to ~ W096/06946 2 1 9 8 4 ~ 9 PCTNS95/10630 solution techniques are: (1) more sensitive (detection of monolayer quantities); (2) easier to separate DNA from sample (avoid interferences); and (3) possibility of ~Ptprt;nn of several different DNA in single analysis, with lnr~l;
probes, e.g., as in sequencing studies.
These lnm;nPccPnt systems are of increasing importance in diagnostics. For example, in U.S. Pat. No.
4,372,745, rham~ m;npscpnt labels are used in ; -applications where the labels are excited into a lllm;nPccpnt state by reaction of the label with H2O2 and an oxalate. In these systems, H2O2 oxidatively converts the oxalate into a high energy derivative, which then excites the label. It is ted, that in principle, the ~Z~2 and an oxalate reaction scheme should work with any lnm;nPRcPnt material that is stable under the nY;~;a;nq conditions of the assay, and can be excited by the high energy oxalate derivative. Unfortunately, this very versatility is a major drawback of the 4,372,745 patent: lack of selectivity or specificity, i.e., typical biological fluids containing the analyte of interest also contain a large number of potentially lllminPcrPnt substances that can cause high ba~ku,Lu~..d levels of lnm;nPc ~I,.e.
Thus a need exists for a solid phase system, e.g., a h;ncPn~nr, and method that (1) provides the neces~aLy specificity absent systems that rely on the H202 and oxalate reaction scheme; and (2) do not depend on solution techniques.
The present invention uv~ the limitations and drawbacks W096/06946 2 1 9 8 4 8 9 PCT~S95/10630 ~
of the prior art.
E Of Invention The present invention provides a biosensor and its use for eleuL.ug~ll~,Gted rhPm;]nm;nPccPnt detection of nucleic acid absorbed to a solid surface via th-e use of ruthenium- and osmiu~ _u.,LGining rhPm;lllm;nPqrPnt labels.
An object of the present invention is to provide a film containing an ~lnm;n--m (III) ~lk~nphiqph~ h~ te layer having metallic Alnmin1lm centers, i.e., ionic Alnm;nllm Al(III) centers, for bonding to single-strand or double-strand DNA ; ~ ili7P~ to said aluminum centers. The Alllm;nl-m (III) kAnPh;~ n~ Ate can be provided as a hi n~ncnr having a coating of A~(C4BP) to bond to SS-DNA or ds-DNA.
A further object of the present invention is to provide a h;o~Pnen~ in the form of chips or ele~L.odes with adsorbed DNA that is labelled with a l~minpcopnt label, such as an osmium or rutheniu~ moiety.
A still further object of the present invention i8 to prepare a biosensor by treating a silicon wafer to form a UilL~ ;nm layer and ju~LGposed gold layer, then contacting t_e layered wafer with an anchoring agent; and subsequently immersing the product in Al(NO3)3, hiqphncrhrnic acid (H2O3P(CHI)~PO3H2~ and Al(NO3)3 aqueous solutions.
Another object of the present invention involves the detection of a nucleic acid by lAhPll ;ng with luminescent metal chelates.
~ W096/06946 PCT~S95/10630 21 ~8489 A further object of the present invention is to apply ele~L.~gel.~L~ted ~h~mi ltlm;np~c~nt techniques to a plurality, i.e., arrayed, oligonucleotide probes.
These and other objects will become more ~aL-L
from the following ~tA;l~ description and drawings.
Brief Descri~tion Of The Drawin~c Fig. 1 shows a schematic representation of the silicon electrode of the present invention containing ionic aluminum Al(III) sites.
Fig. 2 shows ; ;l;7ation of ds-DNA on a All(C4BP) film and interaction of Ru(phen)33~ with the ds-DNA.
Figs. 3A-3C show first tA), second (B), and third (C) scans. ECL ~m;~j n~-potential transients at the Al2(C,BP)/DNA-Ru(phen)33+
electrode in 0.19 M phosphate bu;~er, pH
7, containing 0.13 M TPrA. (Sc~nn;nq was halted and the soll~ti~n stirred after each scan.) Scans were initiated at 0 V and were directed toward more positive potentials. Scan rate, 50 mVts.
Figs. 4A-4C show schematic representations of i -~il;7~tion of ss-DNA tagged with Ru~bpy)33~ on the film (Fig. 4A);
immobilization of ss-DNA on the film and hybridization of complementary strand DNA
W096io6946 2 1 9 8 4 8 9 PCT~S95110630 ~
tagged with Ru(bpy)3~+ (Fig. 4B);
i hi li7~tion of poly(dA) on the film, hybridization of poly(dT), and then interaction of Ru(phen) 32+ with the ds-DNA(poly(dA)-poly(dT)) where the -r~pL~senL~ an ECL active species.
Figures 5A-5C show a cyclic vnl ~ (Fig. 5A);
an Pm1qcinn-potential transient of the Al~(C4BP)/~-l ss-DNA-Ru(bpy)32+ electrode in 0.19 M phosphate buffer/0.13 M TPrA, pH 7 (Fig. 5B); and an pmi ~i nn-potential transient of the Al2(C4BP)~-l ss-DNA in the same solution (Fig. 5C). The electrode used in the experiments shown in Figures 5A-5C wa6 ~,~paL~d as described in the ~p~~ifir~tion with immersion in a 1.38 ~M
~-1 ss-DNA-~u(bpy)32+ or ~-1 ss-DNA
solution for -4 h, respectively. In each case, the potential was scanned from 0 to 1.60 V at v = 50 mV/s.
Figure 6 shows an emission-time transient for the Al2(c~sp) /A-l ss-DNA-Ru(bpy)33+ electrode in 0.19 M phosphate buffer/0.13 M TPrA, pH 7, when the potential was stepped from 0 to 1.5 V.
Figures 7A and 7B show ECL pmi q5io~-potential transients at the Al2(C~BP)/~-lc ss-DNA/A-1 ~ W096106946 PCT~S9~10630 2t ~8~8~
ss-DNA-Ru(bpy)32~ electrode (Fig. 7A) and at the Al2(C4BP)/~-l ss-DNA/~-l ss-DNA-Ru(bpy)32~ electrode (Fig. 7B) where both electrodes are immersed in O.l9 M
phosphate buffer, pH 7, containing 0.13 TPrA and the potential was scanned from O
to 1.60 at scan rate, 50 Nv/s.
Figures 8A and 8B show ECL emission-potentlal transients at the Al2(C4BP)/poly(dA)/poly(dT)/Ru(phen)32+
electrode (Fig. 8A) and at the Al2(C~BP)/poly(dA)/Ru(phen)32~ electrode (Fig. 8B) where both ele~L,udes are immersed in O.l9 M phosphate buffer, pH 7, cnnt~in;nq 0.13 M TPrA, and the potential was scanned from O to 1.60 at scan rate, so mV/s.
Figures 9A-sC show TEM images of Au substrate coated on a Formvar film on a ~400 Cu grid (Fig.
9A); the Al2(C4BP) film on the Au substrate (Fig. 9B); i -'-il;7ed calf thymus ds-DNA
on the Al2(C4BP) film, prepared by immersing the film in a 1.65 mM [NP] of ds-DNA solution for - 4 h (Fig. sC).
Figures lOA-lOC show TEN images of the Al2(C~BP) film on Au substrate coated on a Formvar film (Fig. lOA); immobilized calf thymus W096/06946 PCT~S95110630 ~
21 984P~9 ds-DNA on the Al2(C4BP) film, prepared by immersing the film in a 1.65 mM [NP] o_ ds-DNA solution, for -4 h (Fig. lOB);
immobilized sonicated calf thymus ds-DNA
on the Al2(C4BP) film, ~L~al~d by immersing the film in a 1.65 mM [NP] of ds-DNA solution, sonicated for ~6 h at room-temperature, for -4 h. (Fig. lOC) Figures llA - llB show sequencing via array hybridization.
Det~ilp~ Descri~tion Of Invention m e present invention relates to a sensor and method of dPt.~t;n~ nucleic acids using the sensor. The sensor can be a chip, electrode, or an appropriately --~if;PS surface _or adsorbing ss-DNA or ds-DNA. ~he nucleic acids detected by the method of the present invention include DNA, cDNA or any synthetic variant thereof. A nucleic acid as used thL uu~houL
the spp~i f ~ cation and in the claims is meant DNA or any synthetic variant thereof.
Examples of DNAs detectable by the present method include ~hLI ~ DNA, plasmid DNA, viral DNA, bacterial DNA
and rec~hin~nt DNA. The length of nucleic acid sequence capable of ~PtPctinn by the present method ranges from about 2.7 nm to about 200 nm. In a preferred P~hc~;- L, the nucleic acid sequence ranges from 8 base pair (bp) nucleotides to 3,000 base pair nucleotides. In a most ~ W096/06946 2 I q 8 4 8 9 PCT~S95/10630 preferred ~ L ranges from about 30 bp nucleotides to 1,500 bp nucleotides.
In the present invention, the nucleic acid ce~
to be ~ L~cl may be of purified nucleic acid or may be present in a biological sample. Biological samples in which nucleic acids can be detected using the method of the present invention include but are not limited to biological fluids, e.g., serum, saliva, hair, skin, etc. Alternatively, the nucleic acid can be purified from a sample using methods known to those skilled in the art (Current Protocols in Electrobiology ~l994 ed. Ausubel, F.N. et al. John h7iley &
Sons, Inc.~).
The aluminum (III! Al~n~hicrhrsrhrn~te preferably used is a Al2(C4BP), also [Al2C4BP], film bearing birc~ncAr and is ~repaIed as follows. Silicon wafers were soaked in trichloroethylene for 30 min, rinsed twice with 2-propanol, rinsed with excess amount of d~irni7ed water, and then dried with a stream of dry nitrogen. The clean silicon wafers were primed with a 50 A ~, ;nm layer followed by deposition of a 2000 A gold layer. Chromium and gold targets (99.999%) were used to sputter the films onto the wafers in a MRC Model 8620 system at 10-2 torr. Other terhniqnrc~ such as rh~r;c~l vapor deposition (CVD) to apply the Au or Cr layers, can also be used.
The gold surface supported on the silicon wafers was cleaned with hot chromic acid (saturated R2Cr207 in 90% H2SO~) for -lO s and then rinsed with copious amounts of water. This _g_ W096/06946 r~ o ~
~t q848~
process was repeated until the surface contact angle with water was less than 15~. The clean Au surface was then immediately soaked in an anchoring agent, 0.5 mM 4-mercaptobutylrhn~rhnn;c acid (B PA) solution in absolute ethanol for -24 h. See Fig. 1. The rhnsrhnric acid terminated surface was then thoroughly rinsed with the ethanol, dried with a stream of N2 and then ; ~ed alternately in S ~M Al(N03)3, 5 mM bisphosphonic acid (H203P(CH2)4pO3H2) and 5 mM Al(N03)3 aqueous solution, taking -4 h for each immersion, with washing with water between each step.
Although the preferred pmho~; L contemplates the use of Al centers, other metzl centers, such as, lanthanum (La) and zirconium (Zr) are also rnntP-rl~ted. In addition, although a C4 specie was utilized in producing the ~lk~npb;~l~h~ t~
film, other chain links, C2-C~ may be nt;l;7~, which result in adequate spacing of the ~ m;m~m ions on the surface of the film to permit contact with the phosphates of the DNA
h~rkhnnP. In other words, the "spacer" may range from 2 to 16 carbons in length.
The sensor described above is used by:
a) adsorbing a nucleic acid onto a film containing metal centers;
b) reacting nucleic acid adsorbed to said film with a lnminp~cpnt metal label; and c) detecting the nucleic acid metal label chelates formed in step b) via electrogenerated chemilllm;npsc~nre of said chelates.
~ WO 9610C946 2 ~ 9 8 4 8 9 PCT/USgS/10630 In step (a), the film to which the nucleic acid is adsorbed should contain metal ions which are suitably spaced on the surface of the film to allow interaction of the metal with the phosphate hArkh~nP of the nucleic acid sequence.
r 1P~ of metal centers suitable for use in binding to nucleic acid phosphate groups are Alllmimlm, lAnthAnl.m, and zirconium. In a preferred s ~i nt, the film c~nt~;nq an Alllmir~lm Al (III) metal center.
The nucleic acid adsorbed to the film in step (a) of the method of the present invention may be either double-stranded or singlc ~LL~I.ded. When a single-stranded nucleic acid is AA~rhed to the film, the adsorbed single-stranded nucleic acid is then hybridized to a 1 y single-stranded nucleic acid sequence. Conditions of hybridization are ut;1i~r~ which promote base pairing between the single-aLL-nded DNA adsorbed to the film in its complementary se~uel,c~. Factors influPn~ing hybridization between nucleic acid sequences are known to those skilled in the art and include salt uu..~enLL~tion of the hybridization solution, hybridization temperature and stringency of post-hybridization washes. In addition, the length of hybridization may also be controlled to optimize binding. Satiable buffers in which to carry out the hybridization reaction include 5mN Tris buffer, p~ 7 containing 50 mM NaCl.
The . 1 Lary single-stranded nucleic acid sequence hybridized with the nucleic acid adsorbed to the film may be nnlAhpled or labeled with a luminP-cPnt metal label.
W096/06946 l~IIU~ o ~
Suitable ln=inpccpnt labels include ruthenium- and osmium-containing labels where ruthenium or osmium are bound to at least one polydentate ligand. If the metal has greater than one polydentate ligand, the polydentate ligands may be the same or different. (Other known ECL active labels can also be utili7P~ that emit at different wave lengths such as organic ECL labels, e.g. sulfonated-9,lo-diphenylanthracene.) Polyde.,Late ligands of either ruthenium or osmium include aromatic and aliphatic ligands. Suitable aromatic polydentate ligands include aromatic het~Lu~yclic ligands. Preferred aromatic heterocyclic ligands are nitrogen-containing, such as, for example, bipyridyl, bipyrazyl, terpyridyl, and phPn~ L1" ulyl. If the metal chelate has greater than one polyd~..L~Le ligand, the polydentate ligands may be the same or different.
Suitable polydentate ligands may be unsubstituted, or substituted by any of a large number of substituents known to the art. Suitable substituents include ~or example, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, carboxylate, car~Y~l~Phyde, carbn~m;dP, cyano, amino, hydroxy, imino, IIYdLU~Y~L~U~IY1~ aminocarbonYl, amidine, gl~ni~;niu=, ureide, sulf~L-~Jntdining groups, phOa~uhuluus containing groups, and the carboxylate ester of N-lly~lLu~y~ rrinim;tl~.
The ruthenium or osmium may have one or more monodentate ligands, a wide variety of which are known to the art. Suitable monodentate ligands include, for example, ~ W096/06946 2 t 9 8 ~ 8 9 ~ ,3,l. ~0 carbon - ~P, cyanides, isocyanides, halides, and aliphatic, aromatic and heterocyclic phosphines, amines, stibines, and arsines. A more complete list of the ligands, - e.g., = ' L~Le and polydentate ligands, that can be used in the present invention are set forth in U.S. Patent Nos.
5,310,687, 5,Z38,808 and 5,221,605, the subject matter of which are in~L~uuL~ted herein by reference.
It is also within the scope of this invention for one or more of the ligands of the metal to be attached to additional rh~mirAl labels, such as, for example, rA~;oArr;ve isotopes, fluorescent _ ~-, or additional lnr;neqcpnt ruthenium- or osmium-containing centers.
The compl~ L~LY single-stranded nucleic acid may be tagged with the preferred lnminpccpnt metal labels of the present invention via co-valent bonding to one or more of the polydentate ligands of the metal label through one more amide linkAqP~. This linkage may be oriented so that the nucleic acid is bonded directly either to the carbinol or to the nitrogen of the amide linkage. These rhPrirAl moieties may be ionized. A more elaborate description of methods for attaching a l llr; nPcrPnt ruthenium-containing or osmium-containing label to amino groups of biological substances such as nucleic acid is provided in U.S. Patent No. 5,Z21,605 incorporated by reference.
In an alternative '--';r L, the complementary single-stranded nucleic acid is unlabeled and hence, hybridization to a single-stranded nucleic acid adsorbed to a w096/06946 2 ~ 9 8 4 8 ~ ~ l/U ~ 0 ~
film results in generation of nnl Ahpl ed double-stranded nucleic acid ~crrbpd to the film. Thus, double-stranded nucleic acid can be adsorbed to the film directly or can be created by first adsorbing single-stranded nucleic acid to the film and then hybridizing the adsorbed nucleic acid with its compl b~Ly se~uence. In either case, the film rnnt~;n;nq the ~scnrhed doublc ~L~ended nucleic acid is then immersed in a solution containing lnm;npcrpnt metal label or a solution suitable for promoting intercalation of the metal with the double-stranded nucleic acid. Examples of suitable solutions include, but are not limited to water.
A nucleic acid in which the lnm;nPecPnt metal containing labels intercalates to produce nucleic acid--metal label chelates is then detected by ; n~nr; nq the metal label present in the chelates to emit ele~Ll, L-n radiation by creatiny an excited state of the metal species that will lnm;nP~re at wave lengths from about 200 nanometers to about 900 nanometers, at am~ient temperatUres. Intercalation (or more generally, association) of the ECL labeled species with DNA depends upon the experimental conditions in which the label is partially inserted between the base pairs of DNA. It is considered "association" because of an ele~LLvaL~tic interaction between a positively-charged label and the negatively-charged phosphate groups on the DNA. The exact nature of the interaction of Ru(phen)32~ with DNA is uncertain, but is believed to be intercalation. The temperature must be below the melting point of ds-DNA, preferably about 25-30~ C.
-~4-~ W096/06946 2 ~ 9 8 4 ~ 9 PCT~S9~10630 The pH is typically near 7, but within a range of about S to about 8. The intercalation or association reaction must be given s~lfC;ciPnt time to occur; about 30 to about 60 minutes, although times as short as 10 minutes also work.
In one omho~i L of the present invention, the metal label is excited by exposiny the nucleic acid-metal label chelates to electrorhnmic~l energy. The potential at which the oxidation of the metal label will occur depends upon the exact structure of the metal label as well as factors such as the co-reactant utilized, the pH of the solution and the nature of the electrode used. Examples of suitable co-reactants which, when incubated with the nucleic acid-metal label chelates in the presence of the ele~LLv~h~;c~l energy, will result in Pm;~s;on of the metal label intercalated with the nucleic acid, include tripropylamine (TPrA), oxalate or other organic acid such as pyruvate, lactate, malonate, tartrate and citrate. This oxidation can also be performed rhPmir~lly, with some strong oxidants such as PbO2 or a Ce(rV) salt.
Those of ordinary skill in the art rprogn;7e how to determine the optimal potential and emission wave length of an electrochemilnminPcrPnt system. The electrorhPmiluminPqrPnt species may be measured by any suitable r- ' ~n;~m such as measurement of an electric current or emitted electromagnetic radiation. For example, the rate of energy inputted into the system can provide a measure of the lnminpcr-pnt species.
Suitable measurements include, for example, mea~ul~ Ls of W096l06946 2 1 98489 . ~ o ~
electric current when the lnminpcrpnt species is generated electrorhPm;c~lly, the rate of reductant or oxidant utilization when the lllm;npccpnt species i5 generated rhPm;r~lly or the absorption of ele~L-~ ~tic energy in S photolnminpcrpnt techniques. In addition, of course, the lllminPcrPmt species can be detected by measuring the emitted ele~Lr~ ~ Lic radiation. All of these measu., L~ can be made either as continuous, rate-based mea~u., L~, or as cumulative methods which ~ te the signal over a long period of time. An example of rate-based mea~uL~ Ls would be by using photomultiplier tubes, photodiodes or phototransistors to produce electric currents proportional in magnitude to the incident light inten_ity. r lpc of cumulative methods are the integration of rate-based data, and the use of photographic film to provide cumulative data directly.
All of these lllminperpnre-based methods entail repeated lnminPccPnre by the ruthenium-containing _ '.
The repetitive nature of the detectable event distinguishes these labels from radioactive isotopes or bound rhPmilnminPcrpnt molecules such as luminol. The latter labels produce a detectable event only once per molecule (or atom) of label, thereby limitiny their detectability.
The following examples illustrate various aspects of the invention but are in no way intended to limit the scope thereof. Analysis was performed in a Plexiglas cell ~PcirnpA
for ECL and electrochemical studies using the film of the wos6/u6s46 PCT~S9~H0630 2~ 98489 present invention, a gold surface supported on the silicon wafer containing r 'allic sites, e.g., Al. A s~uL~ted calomel reference electrode (SCE) and a platinum wire counter electrode were used for all nea~uL~ L8. The electrorhpm;
mea~uL~ Ls coupled with ECL experiments were carried out with a Nodel 175 universal ~LOU~L ~ a Model 173 potentiostat (Princeton Applied r ~ , PAR, Princeton, NJ), and an Ominigraphic 2000 X-Y recorder (Houston In~LL~ n~c, Houston, TX). The ECL emission was detected by a Model C123 single-photu.l cuurlLiny system (Hamamatsu Corp., Bridgewater, NJ) utilizing a Hamamatsu R928P PMT, cooled to -20~C in a Model TE 308 TSRF cooler controller (Products for Research Inc., Danvers, MA). The meter output was fed into the y-axis of the x-y 1~CULd~L, and the signal from the potentiostat was fed into the x-axis to afford ECL intensity versus bias potential display. Solution analysis by ECL was carried out uith a QPCR analyzer (Perkin-Elmer, Norwalk, CT).
A MRC (Materials Research Corporation, Orangeburg, NY) Model 8620 sputtering system at 10-2 torr, with an RF power of 150 W and RF peak to peak voltage of 1.8 KV, was used to sputter gold (99.999~) on silicon wafers.
Polydeoxyadenylic acid (Poly(dA)), polythymidylic acid (poly(dT), polydeoxycytidylic acid (poly(dC)) and calf thymus (CT) ss-DNA and ds-DNA were obtained from Sigma ch~ l Co. (St. Louis, MO) and were used without additional purification. The ss-DNA samples, ~-1 DNA
(5'GAAAATGTGCTGACCGGACATGAA~ATGAG3'), (Seq. ID. No. 1) ~ DNA
W096/06946 2 1 9 8 4 8 9 PCT~595110630 tagged with Ru(bpy)32+(5'Ru(bpy)32+-GAAAATGTGCT~-~CCGc-~c~GAAAATGAG3~), (Seg. ID. No. 2) and a comp]~ ~L~ strand ~-lc DNA
(5'CTCATTTTCA~lCCG~cAGCACATTTTC3'), were obtained from Perkin-Elmer and diluted with a 5 mM tris buffer containing 50 m~ NaCl(p~ 7). Synthesis of ss-DNA can be carried out on a DNA synthesizer (e.g. Applied Biosystems, Model 381A). See also L.J. NcBride and M.H. Cruthers, Tetrahedron Letters, 24, 245 (1983).
The reagents used in the following examples include trichloroethylene (99.6%), 2-propanol (99.9~), tripropylamine (TPrA) (98%), Ru(bpy)3Cl2.6H2O, Ru(phen)3Cl2O, ethyl alcohol (200 proof), Al(NO3)3.9H2O, KICr20~, NaHzPO~ and tris ~hydL uxy ' hyl ) 15 ~mir ~'-n~ and were used as received without purification.
Ri ~I.Iln~.l.. i o acid H2o3p(cH2)4po3H2 (C~BPA)~ and 4 mercaptobutylph~,lh i~ acid (MBPA~ were synthesized in a~L~ e with the techniques taught by Mallouk et al, J.A.C.S., 115, 11855 (1993). The assay buffer for ECL
experiments contained 0.13 M TPrA and 0.19 M phosphate buffer, ple~aled by dissolving TPrA into a NaH2PO~ 601ution and adjusting the pH to 7 with 1 M NaOH.
Deinni~od water from a Millipore Milli-Q (18 Mn-cm) system was used to prepare all aqueous solutions and to rinse the electrode surface.
TEN 8_mple PreparAtion.
TEM samples were prepared by coating Au on a Formvar film on a #400 Cu grid with a vacum evaporator (Edwards 306), ~ W096/06946 2 1 9 8 4 8 9 ~ oo~
fabricating the All(C4BP) film on Au following the procedure described above and then ; h;l;7ing DNA on the Al2(C4BP) film. A trAn~ si~ electron microscope tJE0L lOOCX) at 80 KV was used to image the Au substrate, the Al2(C4BP) film and the i -'-ili7e~ DNA.
W096/06946 2 1 9 8 4 8 ~ PCT~S9~/10630 r~m~le 1 Flectro~eneratea rhp~ eqc~nt Detection of sd do-3NA l-q;nrt A RUthenium L~bel.
Calf thymus ds-DNA was ; -hi 1; zed on the surface of an All(C~BP) film by immersing the film in a solution of DNA
(1.9 m~ in nucleotide phosphate, NP) for 4 h (Figs. 1 and 2).
The film was then rinsed three times with 4-mL portions of ~r;nn;7n5 water and then immersed ;or 4 h in either an agueous 0.56 m~ Ru(phen)3Cl2 solution or a 0.12 mM Ru(phen)3(Cl0~)2 solution in MeCN. Ru(phen)32+ associates with ds-DNA and can be detected through its ele~Lluy~neLaLed chemilllm;n~6c~nre (ECL). Alternatively, the film could be soaked in a mixed ds-DNA (1.9 mM NP) and Ru(phen)3Cl2 (0.12mM) solution for 4 h to produce the ada~LBed layer.
ECL was produced by scanning the potential of the electrode following film formation, DNA adsorption, and Ru(phen)33+ association, from 0 to 1.6 V vs. a saturated calomel electrode (SCE) while it was ; aed in a solution of 0.19 M phosphate buffer (pH 7) containing 0.13 M tri-n-propylamine (TPrA). Typical ECL transients, detected with a single-photon-counting apparatus, are shown in Figures 3A-3C.
Emission arises from the energetic electron-transfer reaction between ele.LLogenerated Ru(phen)33+ and an interm-ediate in the oxidation of TPrA:
DNA-Ru(phen)32+ (ads) - e - DNA-Ru(phen)33+ [1]
CH3CH2CX2-N-Pr2 - e - CHlCH~CHINPrl+ [2]
CH3CHICH.NPr2+ - CH3CH,CHNPr2 + H+ [3]
~ W096/06946 2 ~ 984 89 r~ r DNA-Ru(phen)33+ + CH3CH2CHNPrllDNA-Ru(phen)32++r~,r~,r~NPr2+ [4]
DNA-Ru(phen)32+ ~ DNA-Ru(phen)32+ + hu [5]
The Pm;~ion intensity decreased on a second scan and none was seen on a third, suggesting 1058 of Ru(phen)33+ from the film and diffusion into the bulk solution.
In a control experiment, an electrode with a film of Al2(C4BP) that had not been treated with DNA was soaked for 4 h in either an aqueous 0.56 mN Ru(phen)32+ or a 0.12 mM
Ru(phen)32+ in NeCN solution and then rinsed with NeCN and water. This electrode showed no ECL emission upon potential sweep in the same TPrA solution described above, d~ LL~ting the adsorption of Ru(phen)32+ on the Al2(C4BP) film does not occur. The ability to generate Ru(phen)33+ electrorh~mirAlly in this ECL experiment ~ L~tes that the Al2(C4BP) film and the DNA layer do not prevent het lugeneuus electron-transfer reactions.
To obtain further evidence for Al2(C~BP) film formation and DNA immobilization, experiments were undcrtaken with a gold-coated quartz crystal and a quartz crystal micrnh~l~nre (QCX). The gold was repeatedly treated with hot chromic acid and then rinsed with water and EtOH until the surface was hydrophilic, as indicated by contact angle mea~ur~ L~. The crystal frequency was then measured in air during different stages of Al~(C4BP) film formation, after DNA
adsorption, and after Ru(phen)32+ association. The film was rinsed after each step with deionized water and dried in a W096/06946 2 t ~ 8 4 8 ~ F~l/u~ ~10630 stream of N2 before measurement of the frequency. Results are shown in Table 1.
~ W096/06946 2 1 9 8 4 8 9 PCT~S95110630 T~bln l. F~ ~c~ and Ma~4s Changes on a QCH Plato for Fil_ Growth. DNA I_mobiliz~tion, ~nd Ru~Phen)3l+ Bindinq.
After immersion in: ' ~f~ (Hz) ~m (ns) 109rh (mol/cm NBPA~ -70 137 Al(NO3)3' -27 53 l' C4BPAd -145 283 2 Al(NO3)1' -25 49 l~
ds-DNA' -24 47 0.3i Ru(~hen)~2+~ -13 35 0.l 10 a Immersions were sequential in order given from top to botto_;
bare surface was gold; bare crystal freguency, 6.011329 MHz.
0.5 mM M~3PA in EtOH for - 24 h.
' 5 mM ag. Al(NO3)3 for - 4 h.
d 5 mM ph~1h--~ic acid for 4 h.
' Calf thymus DNA (3.8 mM NP) for 4 h.
f 0.24 mM Ru(phen)32+ in MeCN for 4 h.
The variAtion and drift of the signal over the series of mea~u~ L~ was about + l Hz.
h Ac51lming ro--ghnPcs factor for gold of 2 (total surface area, 0.6 cm2). For comparison, for a close-packed monolayer of MBPA, r = o . 6 x 109 mol/cm2.
i Acc11ming an Al(H2O)33+ until adsorbed.
i Cur~ 7;nq to moles of nucleotide phosphate (NP) per cm2.
- Clearly, the crystal fre~uency decreases as the Al2(C4i3P) film forms and DNA and Ru(phen)32+ are adsorbed on the surface, showing an increase of mass on the crystal during the different stages. The mass change, ~m, can be related to the W096/06946 ~ 2 1 984 8~ P~ oo~o ~
frequency change, ~f, by the Sauerbrey equation:
Qm = -(A ~ /2Fo2)~f where Fo i5 the fnn~ 1 fL~uu~ y of the llnloA~Pd crystal (6 MHz), A is the electrode area (0.159 cm2), pq is the density~
of quartz (2.65 g/cm3) and ~q is the shear modulus of quartz (2.95 x 101l dyne/cm2). With these constants, ~m (ng) = -1.95 ~f (Hz) The mass changes calculated in this way are also given in Table 1. These can be cu-lv~LL~d to approximate surface o ~u~ .LLaLions, r, ACcllming a Luu~l.ne_s factor of 2 (i.e., total surface area of both sides of the quartz crystal of 0.6 cm2). ECL could also be detected from both gold surfaces of the ~uartz crystal, when used as a ~ub~LL~te for Al2(C4BP) film formation, DNA adsorption and ~u(phen)3l+ association and then ~5 scanned in the TPrA solution.
The electrode surface can be designed with immobilized DNA without adsorbing a detector molecule, Ru(phen)32~. A ds-DNA on the surface can be detected by eleu ~ uu~n~L~ted ~hpm;lllm;npcrpn~e of associated Ru(phen)3t~.
Single-stranded DNA can also be i ~;1;7ed on the Alt(C4BP) film surface and then hybridized with complementary DNA in solution with detection of the ds-DNA produced by ECL.
~ W096106946 2 1 9 8 4 8 9 PCT~S95/10630 F!v~mnle 2 8i~qlc ~L~ n'~ i7ation and hvbridiz~tion.
~-1 tagged ss-DNA ti.e., labeled with Ru(bpy)32+) was ; h;li70~ on an Alnm;nnm phosphate film of the present invention by immersing the film in the DNA solution tFig. 4A).
The amount of ; -hili7sd DNA-Ru(bpy)32+ on the surface was tprmino~4~ by ECL resulting from the oxidation of Ru(bpy)l2+
and TPrA in a solution.
Untagged ~-lc ss-DNA was ; i1;70~ on an Alllmin~lm phosphate of the present invention. The ~-lc ss-DNA
containing film was incubated in a complementary strand ~-1 tagged ss-DNA solution at 60OC for 5 min and then cooled to room +E , ~LULC gradually; during this cooling the ss-DNA
hybridized with the compl LaLy strand DNA (Fig. 4B). The hybridized DNA-Ru(bpy)32+ on the film was detected by ECL a8 described above.
Poly(dA) was immobilized on an Alllminllm phosphate film of the present invention by soaking the film in a poly(dA) solution. After the ; ~il;7ation, poly(dT) was hybridized with poly(dA) to produce poly(dA)-poly(dT) ds-DNA
on the surface by incubating the film in a poly(dT) solution at 70~C for 5 min and then cooled to the room temperature gradually (Fig. 4C). To intercalate Ru(phen)32+ into the ds-DNA (poly(dA)-poly(dT)), the Al2(C4BP)/poly(dA)-poly(dT) film was treated with a Ru(phen)3Z+ solution. The hybridized poly(dA)-poly(dT)-Ru(phen)32+ on the surface was detommined by ECL based on the oxidation of Ru(phen)32~ and TPrA in the W096/06946 2 t 9 8 ~ 8 9 r~ o solution.
ExamPle 3 ~E ~ili7~tion ana Detection of 30 bp ss-DNA.
- hi 1~ ~ed on A~ m PhosPh~te Film.
The aluminum phosphate film, prepared as described above was immersed in a 1.38 ~M solution of ~-1 30 bp ss-DNA
~tagged with Ru(bpy)32+) for -2 h. This was employed as a working electrode for an ECL experiment in a 0.19 ~ phosphate buffer (pH 7) containing 0.13 M TPrA. Cyclic vol+, , a~
and t~ cion transients were obtained by scanning the potential of the electrode from O to 1.6 V vs, a saturated calomel electrode (SCE). A L~r ese--Lative voll a~ and t~ -;nn detected with a single-photon-counting system are shown in Figures 5A-5C. The broad oxidation was at - 1.2 V
and a small reductlon wave at -0.35 V observed in the voli a~ (Fig. 5A) arise from the oxidation of TPrA, Ru(bpy)3Z+ and the Au substrate and the reduction of the oxide of Au. The ECL P~iccion from the Al2(C~BP)/~-1 ss-DNA-Ru(bpy)3~+ electrode (Fig. 5B) tl Lr aLes that the ~-1 30 bp ss-DNA-Ru(bpy)3l+ was immobilized on the film. The decay of the light intensity (I) as a function of time (t) was also investigated with the single-photon-counting system. The light intensity t1PtPt'tPtl by the photomultiplier tube, when the potential was stepped from O to 1.50 V, decreased with time (Fig. 6). The decay o~ intensity with time suggests desorption of ss-DNA-Ru(bpy)32+ from the electrode surface or ~ W096/06946 2 1 9 8 4 8 9 PCT~S95/10630 irreversible de _sition of the emitter. Emission results from the energetic electron-transfer reaction between electrogenerated Ru(bpy)33+ and an in+~ te in the oxidation of TPrA:
DNA-Ru(bpy)32+ (ads) I DNA-Ru(bpy)33+ + e t6 CH3CH2CH2NPr2 - e - CH3CH2CH2NPr2 t7]
r~,r~ Npr2+ _ r~Jr~lr~Npr2 + H+ [8]
DNA-Ru(bpy)33++CH3CH2CHNPr2 -- DNA-Ru(bpy)32+ +CT~,r~t'TTNPr2+ [9]
DNA-Ru(bpy)32+ - DNA-Ru(bpy)32+ + hu [10 No ECL Pmi~inn was observed from a film prepared by immersing it in solu+inn of llnllhPlpd ss-DNA: 1.38 ~ A-1 30 bp ss-DNA
or a 0.37 ~N A-lc 30 bp ss-DNA snlntinn (Fig. SC).
A control experiment with the aluminum phosphate film i ped in a 304 n~ Ru(bpy)32+ solution for - 4 h shows a negligible ECL signal, indicating that Ru(bpy)32+ does not adsorb on the film and oxidize.
~-mDle 4 Detectio~ of 30 bD ss-DNA Hvbridized to Com~l D ~ 85-DNA Ta~ed ~Lt~ A Ruthenium Label.
After the ; 'il;7ation of nnl~hPl~d A-lc 30 bp ss-DNA on the Al2(C4BP) film, the film was immersed in a 1.38 ~M
compl L~LY strand ss-DNA (tagged with Ru(bpy)32+) (A-l 30 bp ss-DNA-Ru(bpy)32+) solution. The film in the solution was gradually heated to 60~C in water bath, incubated at 60DC for W096/06946 2 1 9 8 4 8 ~ PCT~S9~10630 5 min. and then slowly cooled to room-t~ e~LuLes, during which the ~-1 ss-DNA-Ru(bpy~ 32+ was hybridized with the compl y strand ~-lc ss-DNA on the surface as ill~L,~Led in Fig. 4B. The film with hybridized DNA was employed as the working electrode in an ECL cell as described above. The ECL
emission from the film was observed as shown in Fig. 7A.
However, when ~-1 ss-DNA, rather than ~-lc ss-DNA, was i hili7n~ on the film and followed by the same hybridization ~L u~eduL ~ as described above by incubating the film in the ~-1 ss-DNA-Ru(bpy)32+ solution, heating to 60~C, and then cooliny to roon-temperature, no obvious ECL emission was evident as shown in Fig. 7B. This d -LL~tes that the ECL nmiCR;~n in Fig. 7A arises from the hybridization of ; - ili 7r~r1 ~-DNA with ~-1 ss-DNA-Ru(bpy)32+ in the solution. The nmiqcirn again results from the oxidation of Ru(bpy)32+-tagged hybridized ss-DNA and TPrA in the solution as described by Eqs. [6] - [10]. In this experiment the Al2(C,BP) film should be incubAted in the A-l ss-DNA solution for a sufficiently long time (at least 4 h) to cover all possible Al (III) binding sites on the film with the ~-1 ss-DNA before the Al2(C4BP)/~-1 ss-DNA electrode is exposed to the h-l ss-DNA-Ru(bpy)32+. It is preferred to cover all Al3+ adsorption sites to avoid r~micc;o~ from the film/~-1 ss-DNA/~-1 ss-DNA-Ru(bpy)32+ arising from some immobilization of ss-DNA-Ru(bpy~ 32+ .
W096/069~6 P~llu~ O
2~ 98489 FYAmnle 5 Detection of ~olv(dA) RYbridized to ~olY~dT) l~ein~ a Ruth~nillm T.~h~l .
The Alllm;n--m pl~n,lll Ate film, pL~a~d as described S above, was immersed in a 21 ~M poly(dA) solution for -4 h, then in a 0.24 mM Ru(phen) 3Z+ solution for -4 h. When the Al2(C~BP)/poly(dA)/Ru(phen)32+ film was employed as a working electrode in a solution of 0.19 M phosphate buffer (pH 7) containing 0.13 M TPrA, no ECL emission was observed (Fig.
8A). This is consistent with the lack of association of Ru(phen)32+ with ss-DNA. However, a film of poly(dA) hybridized with only poly(dT) to form ds-DNA did produce ECL.
This film, formed by immersicn in the poly(dA)solution, was incubated in a 38 ~M poly(dT) solution (gradually heated to 70~C in a water bath, incubated at 70~C for S min and then slowly cooled to room temperature). It was then treated with a 0.24 mM Ru(phen~32+ aqueous solution and used as a working electrode in the TPrA-containing phosphate buffer for ECL
investigation. ECL emission was observed as shown in a representative Pmiqeion transient (Fig. 8B), ~ ~L~ting that poly(dT) in the solution hybridized with the poly(dA) on the surface to produce a ds-DNA [poly(dA)-poly(dT] and Ru(phen) 32+ intercalated with the ds-DNA as shown in Fig. 4c.
These examples show that Ru(phen) 32+ only intercalates with ds-DNA tpoly(dA)-poly(dT], but not with ss-DNA ~poly(dA)). Tntercalation of Ru(phen)32+ with ss-DNA and ds-DNA, ECL emission was measured from a 43 nM Ru(phen)32+
W096106946 PCT~S95/10630 2 1 ~848~ --solution, a 43 nM Ru(phen)32~ containing 57 ~M nucleotide phosphate ([NP]) of calf thymus ss-DNA and a 43 nM Ru(phen)37' containing 33 ~M [NP] of calf thymus ds-DNA with a ~PCR
analyzer. Results are given in Table 2, ~ W096l06946 2 1 9 ~ 4 8 ~ PCT~S9s/10630 T~ble 2 ECL Or 801utions o~ 43 nN Ru(phen~32+
~ith ss-DNA ~nd d~-DNA
Solution I II- IIID
Ru(phen) 32+ alone 65 -DNA+Ru(phen) 32+ ds-DNA+Ru(phen) 32+
Conc. NP(~M) ~ 57 33 ECL(cts/s) 10068_23 10052_12 7779+268 ~ Ratio of moles of nucleotide phosphate (NP) of ss-DNA to Ru(phen) 32+ = 1 . 3 x 10~
b Ratio of moles of nucleotide phosphate tNP) of ds-DNA to Ru(phen) 32+ = 7.7 x 103 indicating that the ECL emission decreased after ds-DNA was added to the Ru(phen)32+ solution. A negligible change, however, in the ECL signal was shown when ss-DNA was added, further ~ LLGting that Ru~phen)32+ only intercalates with ds-DNA, but not with ss-DNA.
A control experiment in which a film, after the immobilization of poly(dA), was incubated in a 21 ~M poly(dA) or a 46 ~ poly(dC) solution instead of poly(dT) (70OC for 5 min then cooled to room-t G~ULe) and then treated with Ru(phen)32+ for -2 h ~Luduce5 no ECL P~iqSin~. This indicates that the Al2(C~BP)/poly(dA) electrode can distinguish a complementary strand of poly(dT) DNA from non-complementary ones.
W096/06946 21 984 89 ~ r ~Q
~Y~le 6 TE~ Im~ges of the film and calf thymu5 ds-DNA.
Samples, prepared by coating Au on a Formvar film on a #400 Cu grid with a vzcuum evaporator, fabricating the ~ m;ml~ phosphate film on the Au as described above, and then ; 'il;7ing DNA on the A12(C,BP) film by immersing the film in a 1.65 mM ~NP] of calf thymus ds-DNA for 4 h, were imaged with a tr~nC~;cc;~n electron mi~L~s~u~e tTEM) at 80 RV. As shown in Figs. 9A-9C the featureless Au substrate (Fig. 9A) shows formation of crystalline islands of Al~(C4BP) film (Fig. 9B) and clumps of DNA (Fig. 9C). The film (Fig. lOA) was then treated with either 1.6~ mM [NP] calf thymus ds-DNA (Fig. lOB) or an identical solution of ds-DNA that had been subjected to sonication for 6 h (Fig. lOC). The results indicate that smaller clumps of DNA are adsorbed on the film after sonication.
A further ;- ~ of the invention i5 shown in Figs. llA and llB. A sensor surface having a multilayer film with bonding groups is provided with a complete set o~
olign~rleotide probes using similar techniques described above for adsorbing ds-DNA and for SS-DNA. See Fig. llA. The sensor surface of Fig. llA is contacted with DNA to be sequenced. The above ~;crlocP~ ECL yLuceduL~s are then used to recognize zones with a complementary sequence. (Fiq. llB).
When chips are used, different types of ss-DNA that make up a test sequence are attached to the surface of a chip to make an array. The chip array is then exposed to the WO 96/06946 2 1 9 8 4 8 q . ~ 10630 sa~ple solution to be sequenced, with formation of ds-DNA at the appropriate location being recognized by the ECL approach.
Although the invention has been described in conjunction with the specific ~ ~nts, it is evident that many alternatives and variations will be apparent to those skilled in the art in light of the foregoing description.
Accordingly, the invention is intpn~p~ to embrace all of the alternatives and variations that fall within the spirit and scope of the AprPn~P~ claims.
WO 96/06946 2 ~ 9 8 4 8 9 . ~ 'Q
~yU~N~ LISTING
(l) GEh-ERAL INFORMATION:
(i) APPLICANTS: Allen J. Lard and Xia-Hong Hu ~ii) TITLE OF THE INVENTION: 3IOSENSOR FOR AND
METHOD OF ELEcTRor~RNRR~TRn cHE~TTlnMTNRqcENT
D~ 1UN aE NUCLEIC ACID ADSORDED TO A SOLID
SURFACE
(iii) NUM8ER OF 9~yu~Nu~S: 3 (iv) CU~i~UN~NU~ ADDRESS:
(A) ~ : Morgan i~ Finnegan (B) T'.EET: 1299 Pennsylvania Ave., N.W., .u.te 960 (C) ~I'Y: ~ashington (D) ~T.TE: Distrlct of rOl ~'A
(E) I~ CODE: 20004 (v) COMPUTER RR~n~rR FOR~-(A) MEDIUM TYPE: Disket~e, 3.50 inch, 1.44 Mbstorage (B) COMPUTER: I;3M COMPATIPLE
(C) OPERATING SYSTEM: MS-DOS 5.0 (D) SOFTWARE: WordPer~ect 5.1 (vi) CURRENT APPLICATION DATA-(A) APPLICATION NUM~ER: 08/296,630 (E) FILING DATE: 08/26/94 (C~ CLASSIFICATION: N/A
(vii) PRIOR AppT~Tr~TTnN DATA: N/A
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Michael S. Marcus (B) REGISTRATI0N NUMBER: 31,727 (C) R~5K~N(~;/DOCXET NI~BER: 23247001 ~ix) TRTRcnu~TNTr~TTnN INFORMATION:
(A) TELEPHONE: (202) 857-7887 (B) TELEFAX: (202) 857-7929 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENC. r~R~r~RRT.CTICS:
(A) LE-GTH: 30 (~) TY E: nucleic acid (C) S~ RNnRnNRqS: single ~D) TO OLOGY: linear ~ W096/06946 2 1 9 8 4 8 9 PCT~S95/10630 (ii) MOLECULE TYPE:
(iii) ~Y~Ol~hllCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: L~3DA
(vi) tlRTr7TN~T SOUR OE :
A) ~t~'TT'qM
'3~ S~ A.N
f, 1 J-.lJJ.L ISOLATE:
D~r:.OP.~NTAL STAGE
' H~'~:,OTY._:
,~, T~.-'UE ~'PE:
C~, C ,, TY:'.':
:: C.,~ LI~.:
_, o r,,NT.~T,, (vii) IMMEDIATE SOURCE
(A) LIBRARY
~nSrRRTn TO A SOLID SURFACE
Field Of Invention The present invention relates to the diagnostic field, and pcppc;Ally nucleic acid diagnostics. In particular, the present invention relates to a probe or sensor having a film containing metal centers, its preparation where a single _LLGnd or double-strand nucleic acid sequence i5 i h;l;7P~ thereon on, and its use in the subsequent detection of the nucleic acid by lAhPl 1 ing with lnm;nPccPnt metal chelates.
In addition to using the subject hi oSPnC~r for nucleic acid diagnostics based on a surface designed modified electrical sensor, i.e., chips or electrode, ; -hili7ation and hybridization of nucleic acid such as DNA on a self-assembled thin film via suriace reaction is also useful in studying molecular recognition of DNA.
Back~round Of Invention Nucleic acid diagnostics has become an important area in molecular biology and biotechnology studies, with applications to the determinations of disease and food contaminating organisms and in forensic and enviLl LG1 investigations. The development of new DNA hiosPnc~rs has led to the application of several detection techniques such as W096/06946 2 1 9 8 4 8 9 PCT~S9~/10630 ~
optical methods (e.g., lnminpccpnre~ Pll;t y and pseudo-Brewster angle rPflectl y), piP7oPlPotric devices (e.g., SAW, QCM), and electrorhpm;rAl technigues (e.g., CV and SWV).
of particular interest are labels which can be made to lllm;npcre through phorrrhPmicnl~ rhPm;c~l, and electrorhDmi~nl reaction schemes. In particular, ele~LLu~l~ ilnm;nPcrPn~
methods of ~Dtprmining the presence of l~hPllD~ materials are preferred over other methods for many reasons. They are highly diagnostic of the presence of a particular label, sensitive, nonhazardous, inpyppnc;ve and can be used in a wide variety of applications. Suitable labels comprise ele~LLu~ llminPcrPnt , inrlll~;nrJ organic and metal chelates.
For example, electrorhpmil~lm;npcrpnt ruthenium- and osmiu~ . I~ininrJ labels have been used in methods for tPrting and guantifying analytes of interest in liguid medin (u~s~ Patent Nos. 5,310,687; 5,238,808; and 5,221,605). In addition, the application of ele~Lu~nerated chemi-lnminPccPnre ~ECL) mea~uL to the detection of solution phase DNA intercalated with ruthenium-containing labels has been described (Carter, M.T. et al. (1990) Bioconiu~ate Chem 2:257-263). However, reaction schemes that are viable in the solution phase are often not applicable in the solid phase.
More importantly, the detection of solution phase analytes such a5 DNA has several drawbacks relative to detection of analytes absorbed to solid surfaces. The advantages for detecting DNA via solid phase techni~ues as opposed to ~ W096/06946 2 1 9 8 4 ~ 9 PCTNS95/10630 solution techniques are: (1) more sensitive (detection of monolayer quantities); (2) easier to separate DNA from sample (avoid interferences); and (3) possibility of ~Ptprt;nn of several different DNA in single analysis, with lnr~l;
probes, e.g., as in sequencing studies.
These lnm;nPccPnt systems are of increasing importance in diagnostics. For example, in U.S. Pat. No.
4,372,745, rham~ m;npscpnt labels are used in ; -applications where the labels are excited into a lllm;nPccpnt state by reaction of the label with H2O2 and an oxalate. In these systems, H2O2 oxidatively converts the oxalate into a high energy derivative, which then excites the label. It is ted, that in principle, the ~Z~2 and an oxalate reaction scheme should work with any lnm;nPRcPnt material that is stable under the nY;~;a;nq conditions of the assay, and can be excited by the high energy oxalate derivative. Unfortunately, this very versatility is a major drawback of the 4,372,745 patent: lack of selectivity or specificity, i.e., typical biological fluids containing the analyte of interest also contain a large number of potentially lllminPcrPnt substances that can cause high ba~ku,Lu~..d levels of lnm;nPc ~I,.e.
Thus a need exists for a solid phase system, e.g., a h;ncPn~nr, and method that (1) provides the neces~aLy specificity absent systems that rely on the H202 and oxalate reaction scheme; and (2) do not depend on solution techniques.
The present invention uv~ the limitations and drawbacks W096/06946 2 1 9 8 4 8 9 PCT~S95/10630 ~
of the prior art.
E Of Invention The present invention provides a biosensor and its use for eleuL.ug~ll~,Gted rhPm;]nm;nPccPnt detection of nucleic acid absorbed to a solid surface via th-e use of ruthenium- and osmiu~ _u.,LGining rhPm;lllm;nPqrPnt labels.
An object of the present invention is to provide a film containing an ~lnm;n--m (III) ~lk~nphiqph~ h~ te layer having metallic Alnmin1lm centers, i.e., ionic Alnm;nllm Al(III) centers, for bonding to single-strand or double-strand DNA ; ~ ili7P~ to said aluminum centers. The Alllm;nl-m (III) kAnPh;~ n~ Ate can be provided as a hi n~ncnr having a coating of A~(C4BP) to bond to SS-DNA or ds-DNA.
A further object of the present invention is to provide a h;o~Pnen~ in the form of chips or ele~L.odes with adsorbed DNA that is labelled with a l~minpcopnt label, such as an osmium or rutheniu~ moiety.
A still further object of the present invention i8 to prepare a biosensor by treating a silicon wafer to form a UilL~ ;nm layer and ju~LGposed gold layer, then contacting t_e layered wafer with an anchoring agent; and subsequently immersing the product in Al(NO3)3, hiqphncrhrnic acid (H2O3P(CHI)~PO3H2~ and Al(NO3)3 aqueous solutions.
Another object of the present invention involves the detection of a nucleic acid by lAhPll ;ng with luminescent metal chelates.
~ W096/06946 PCT~S95/10630 21 ~8489 A further object of the present invention is to apply ele~L.~gel.~L~ted ~h~mi ltlm;np~c~nt techniques to a plurality, i.e., arrayed, oligonucleotide probes.
These and other objects will become more ~aL-L
from the following ~tA;l~ description and drawings.
Brief Descri~tion Of The Drawin~c Fig. 1 shows a schematic representation of the silicon electrode of the present invention containing ionic aluminum Al(III) sites.
Fig. 2 shows ; ;l;7ation of ds-DNA on a All(C4BP) film and interaction of Ru(phen)33~ with the ds-DNA.
Figs. 3A-3C show first tA), second (B), and third (C) scans. ECL ~m;~j n~-potential transients at the Al2(C,BP)/DNA-Ru(phen)33+
electrode in 0.19 M phosphate bu;~er, pH
7, containing 0.13 M TPrA. (Sc~nn;nq was halted and the soll~ti~n stirred after each scan.) Scans were initiated at 0 V and were directed toward more positive potentials. Scan rate, 50 mVts.
Figs. 4A-4C show schematic representations of i -~il;7~tion of ss-DNA tagged with Ru~bpy)33~ on the film (Fig. 4A);
immobilization of ss-DNA on the film and hybridization of complementary strand DNA
W096io6946 2 1 9 8 4 8 9 PCT~S95110630 ~
tagged with Ru(bpy)3~+ (Fig. 4B);
i hi li7~tion of poly(dA) on the film, hybridization of poly(dT), and then interaction of Ru(phen) 32+ with the ds-DNA(poly(dA)-poly(dT)) where the -r~pL~senL~ an ECL active species.
Figures 5A-5C show a cyclic vnl ~ (Fig. 5A);
an Pm1qcinn-potential transient of the Al~(C4BP)/~-l ss-DNA-Ru(bpy)32+ electrode in 0.19 M phosphate buffer/0.13 M TPrA, pH 7 (Fig. 5B); and an pmi ~i nn-potential transient of the Al2(C4BP)~-l ss-DNA in the same solution (Fig. 5C). The electrode used in the experiments shown in Figures 5A-5C wa6 ~,~paL~d as described in the ~p~~ifir~tion with immersion in a 1.38 ~M
~-1 ss-DNA-~u(bpy)32+ or ~-1 ss-DNA
solution for -4 h, respectively. In each case, the potential was scanned from 0 to 1.60 V at v = 50 mV/s.
Figure 6 shows an emission-time transient for the Al2(c~sp) /A-l ss-DNA-Ru(bpy)33+ electrode in 0.19 M phosphate buffer/0.13 M TPrA, pH 7, when the potential was stepped from 0 to 1.5 V.
Figures 7A and 7B show ECL pmi q5io~-potential transients at the Al2(C~BP)/~-lc ss-DNA/A-1 ~ W096106946 PCT~S9~10630 2t ~8~8~
ss-DNA-Ru(bpy)32~ electrode (Fig. 7A) and at the Al2(C4BP)/~-l ss-DNA/~-l ss-DNA-Ru(bpy)32~ electrode (Fig. 7B) where both electrodes are immersed in O.l9 M
phosphate buffer, pH 7, containing 0.13 TPrA and the potential was scanned from O
to 1.60 at scan rate, 50 Nv/s.
Figures 8A and 8B show ECL emission-potentlal transients at the Al2(C4BP)/poly(dA)/poly(dT)/Ru(phen)32+
electrode (Fig. 8A) and at the Al2(C~BP)/poly(dA)/Ru(phen)32~ electrode (Fig. 8B) where both ele~L,udes are immersed in O.l9 M phosphate buffer, pH 7, cnnt~in;nq 0.13 M TPrA, and the potential was scanned from O to 1.60 at scan rate, so mV/s.
Figures 9A-sC show TEM images of Au substrate coated on a Formvar film on a ~400 Cu grid (Fig.
9A); the Al2(C4BP) film on the Au substrate (Fig. 9B); i -'-il;7ed calf thymus ds-DNA
on the Al2(C4BP) film, prepared by immersing the film in a 1.65 mM [NP] of ds-DNA solution for - 4 h (Fig. sC).
Figures lOA-lOC show TEN images of the Al2(C~BP) film on Au substrate coated on a Formvar film (Fig. lOA); immobilized calf thymus W096/06946 PCT~S95110630 ~
21 984P~9 ds-DNA on the Al2(C4BP) film, prepared by immersing the film in a 1.65 mM [NP] o_ ds-DNA solution, for -4 h (Fig. lOB);
immobilized sonicated calf thymus ds-DNA
on the Al2(C4BP) film, ~L~al~d by immersing the film in a 1.65 mM [NP] of ds-DNA solution, sonicated for ~6 h at room-temperature, for -4 h. (Fig. lOC) Figures llA - llB show sequencing via array hybridization.
Det~ilp~ Descri~tion Of Invention m e present invention relates to a sensor and method of dPt.~t;n~ nucleic acids using the sensor. The sensor can be a chip, electrode, or an appropriately --~if;PS surface _or adsorbing ss-DNA or ds-DNA. ~he nucleic acids detected by the method of the present invention include DNA, cDNA or any synthetic variant thereof. A nucleic acid as used thL uu~houL
the spp~i f ~ cation and in the claims is meant DNA or any synthetic variant thereof.
Examples of DNAs detectable by the present method include ~hLI ~ DNA, plasmid DNA, viral DNA, bacterial DNA
and rec~hin~nt DNA. The length of nucleic acid sequence capable of ~PtPctinn by the present method ranges from about 2.7 nm to about 200 nm. In a preferred P~hc~;- L, the nucleic acid sequence ranges from 8 base pair (bp) nucleotides to 3,000 base pair nucleotides. In a most ~ W096/06946 2 I q 8 4 8 9 PCT~S95/10630 preferred ~ L ranges from about 30 bp nucleotides to 1,500 bp nucleotides.
In the present invention, the nucleic acid ce~
to be ~ L~cl may be of purified nucleic acid or may be present in a biological sample. Biological samples in which nucleic acids can be detected using the method of the present invention include but are not limited to biological fluids, e.g., serum, saliva, hair, skin, etc. Alternatively, the nucleic acid can be purified from a sample using methods known to those skilled in the art (Current Protocols in Electrobiology ~l994 ed. Ausubel, F.N. et al. John h7iley &
Sons, Inc.~).
The aluminum (III! Al~n~hicrhrsrhrn~te preferably used is a Al2(C4BP), also [Al2C4BP], film bearing birc~ncAr and is ~repaIed as follows. Silicon wafers were soaked in trichloroethylene for 30 min, rinsed twice with 2-propanol, rinsed with excess amount of d~irni7ed water, and then dried with a stream of dry nitrogen. The clean silicon wafers were primed with a 50 A ~, ;nm layer followed by deposition of a 2000 A gold layer. Chromium and gold targets (99.999%) were used to sputter the films onto the wafers in a MRC Model 8620 system at 10-2 torr. Other terhniqnrc~ such as rh~r;c~l vapor deposition (CVD) to apply the Au or Cr layers, can also be used.
The gold surface supported on the silicon wafers was cleaned with hot chromic acid (saturated R2Cr207 in 90% H2SO~) for -lO s and then rinsed with copious amounts of water. This _g_ W096/06946 r~ o ~
~t q848~
process was repeated until the surface contact angle with water was less than 15~. The clean Au surface was then immediately soaked in an anchoring agent, 0.5 mM 4-mercaptobutylrhn~rhnn;c acid (B PA) solution in absolute ethanol for -24 h. See Fig. 1. The rhnsrhnric acid terminated surface was then thoroughly rinsed with the ethanol, dried with a stream of N2 and then ; ~ed alternately in S ~M Al(N03)3, 5 mM bisphosphonic acid (H203P(CH2)4pO3H2) and 5 mM Al(N03)3 aqueous solution, taking -4 h for each immersion, with washing with water between each step.
Although the preferred pmho~; L contemplates the use of Al centers, other metzl centers, such as, lanthanum (La) and zirconium (Zr) are also rnntP-rl~ted. In addition, although a C4 specie was utilized in producing the ~lk~npb;~l~h~ t~
film, other chain links, C2-C~ may be nt;l;7~, which result in adequate spacing of the ~ m;m~m ions on the surface of the film to permit contact with the phosphates of the DNA
h~rkhnnP. In other words, the "spacer" may range from 2 to 16 carbons in length.
The sensor described above is used by:
a) adsorbing a nucleic acid onto a film containing metal centers;
b) reacting nucleic acid adsorbed to said film with a lnminp~cpnt metal label; and c) detecting the nucleic acid metal label chelates formed in step b) via electrogenerated chemilllm;npsc~nre of said chelates.
~ WO 9610C946 2 ~ 9 8 4 8 9 PCT/USgS/10630 In step (a), the film to which the nucleic acid is adsorbed should contain metal ions which are suitably spaced on the surface of the film to allow interaction of the metal with the phosphate hArkh~nP of the nucleic acid sequence.
r 1P~ of metal centers suitable for use in binding to nucleic acid phosphate groups are Alllmimlm, lAnthAnl.m, and zirconium. In a preferred s ~i nt, the film c~nt~;nq an Alllmir~lm Al (III) metal center.
The nucleic acid adsorbed to the film in step (a) of the method of the present invention may be either double-stranded or singlc ~LL~I.ded. When a single-stranded nucleic acid is AA~rhed to the film, the adsorbed single-stranded nucleic acid is then hybridized to a 1 y single-stranded nucleic acid sequence. Conditions of hybridization are ut;1i~r~ which promote base pairing between the single-aLL-nded DNA adsorbed to the film in its complementary se~uel,c~. Factors influPn~ing hybridization between nucleic acid sequences are known to those skilled in the art and include salt uu..~enLL~tion of the hybridization solution, hybridization temperature and stringency of post-hybridization washes. In addition, the length of hybridization may also be controlled to optimize binding. Satiable buffers in which to carry out the hybridization reaction include 5mN Tris buffer, p~ 7 containing 50 mM NaCl.
The . 1 Lary single-stranded nucleic acid sequence hybridized with the nucleic acid adsorbed to the film may be nnlAhpled or labeled with a luminP-cPnt metal label.
W096/06946 l~IIU~ o ~
Suitable ln=inpccpnt labels include ruthenium- and osmium-containing labels where ruthenium or osmium are bound to at least one polydentate ligand. If the metal has greater than one polydentate ligand, the polydentate ligands may be the same or different. (Other known ECL active labels can also be utili7P~ that emit at different wave lengths such as organic ECL labels, e.g. sulfonated-9,lo-diphenylanthracene.) Polyde.,Late ligands of either ruthenium or osmium include aromatic and aliphatic ligands. Suitable aromatic polydentate ligands include aromatic het~Lu~yclic ligands. Preferred aromatic heterocyclic ligands are nitrogen-containing, such as, for example, bipyridyl, bipyrazyl, terpyridyl, and phPn~ L1" ulyl. If the metal chelate has greater than one polyd~..L~Le ligand, the polydentate ligands may be the same or different.
Suitable polydentate ligands may be unsubstituted, or substituted by any of a large number of substituents known to the art. Suitable substituents include ~or example, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, carboxylate, car~Y~l~Phyde, carbn~m;dP, cyano, amino, hydroxy, imino, IIYdLU~Y~L~U~IY1~ aminocarbonYl, amidine, gl~ni~;niu=, ureide, sulf~L-~Jntdining groups, phOa~uhuluus containing groups, and the carboxylate ester of N-lly~lLu~y~ rrinim;tl~.
The ruthenium or osmium may have one or more monodentate ligands, a wide variety of which are known to the art. Suitable monodentate ligands include, for example, ~ W096/06946 2 t 9 8 ~ 8 9 ~ ,3,l. ~0 carbon - ~P, cyanides, isocyanides, halides, and aliphatic, aromatic and heterocyclic phosphines, amines, stibines, and arsines. A more complete list of the ligands, - e.g., = ' L~Le and polydentate ligands, that can be used in the present invention are set forth in U.S. Patent Nos.
5,310,687, 5,Z38,808 and 5,221,605, the subject matter of which are in~L~uuL~ted herein by reference.
It is also within the scope of this invention for one or more of the ligands of the metal to be attached to additional rh~mirAl labels, such as, for example, rA~;oArr;ve isotopes, fluorescent _ ~-, or additional lnr;neqcpnt ruthenium- or osmium-containing centers.
The compl~ L~LY single-stranded nucleic acid may be tagged with the preferred lnminpccpnt metal labels of the present invention via co-valent bonding to one or more of the polydentate ligands of the metal label through one more amide linkAqP~. This linkage may be oriented so that the nucleic acid is bonded directly either to the carbinol or to the nitrogen of the amide linkage. These rhPrirAl moieties may be ionized. A more elaborate description of methods for attaching a l llr; nPcrPnt ruthenium-containing or osmium-containing label to amino groups of biological substances such as nucleic acid is provided in U.S. Patent No. 5,Z21,605 incorporated by reference.
In an alternative '--';r L, the complementary single-stranded nucleic acid is unlabeled and hence, hybridization to a single-stranded nucleic acid adsorbed to a w096/06946 2 ~ 9 8 4 8 ~ ~ l/U ~ 0 ~
film results in generation of nnl Ahpl ed double-stranded nucleic acid ~crrbpd to the film. Thus, double-stranded nucleic acid can be adsorbed to the film directly or can be created by first adsorbing single-stranded nucleic acid to the film and then hybridizing the adsorbed nucleic acid with its compl b~Ly se~uence. In either case, the film rnnt~;n;nq the ~scnrhed doublc ~L~ended nucleic acid is then immersed in a solution containing lnm;npcrpnt metal label or a solution suitable for promoting intercalation of the metal with the double-stranded nucleic acid. Examples of suitable solutions include, but are not limited to water.
A nucleic acid in which the lnm;nPecPnt metal containing labels intercalates to produce nucleic acid--metal label chelates is then detected by ; n~nr; nq the metal label present in the chelates to emit ele~Ll, L-n radiation by creatiny an excited state of the metal species that will lnm;nP~re at wave lengths from about 200 nanometers to about 900 nanometers, at am~ient temperatUres. Intercalation (or more generally, association) of the ECL labeled species with DNA depends upon the experimental conditions in which the label is partially inserted between the base pairs of DNA. It is considered "association" because of an ele~LLvaL~tic interaction between a positively-charged label and the negatively-charged phosphate groups on the DNA. The exact nature of the interaction of Ru(phen)32~ with DNA is uncertain, but is believed to be intercalation. The temperature must be below the melting point of ds-DNA, preferably about 25-30~ C.
-~4-~ W096/06946 2 ~ 9 8 4 ~ 9 PCT~S9~10630 The pH is typically near 7, but within a range of about S to about 8. The intercalation or association reaction must be given s~lfC;ciPnt time to occur; about 30 to about 60 minutes, although times as short as 10 minutes also work.
In one omho~i L of the present invention, the metal label is excited by exposiny the nucleic acid-metal label chelates to electrorhnmic~l energy. The potential at which the oxidation of the metal label will occur depends upon the exact structure of the metal label as well as factors such as the co-reactant utilized, the pH of the solution and the nature of the electrode used. Examples of suitable co-reactants which, when incubated with the nucleic acid-metal label chelates in the presence of the ele~LLv~h~;c~l energy, will result in Pm;~s;on of the metal label intercalated with the nucleic acid, include tripropylamine (TPrA), oxalate or other organic acid such as pyruvate, lactate, malonate, tartrate and citrate. This oxidation can also be performed rhPmir~lly, with some strong oxidants such as PbO2 or a Ce(rV) salt.
Those of ordinary skill in the art rprogn;7e how to determine the optimal potential and emission wave length of an electrochemilnminPcrPnt system. The electrorhPmiluminPqrPnt species may be measured by any suitable r- ' ~n;~m such as measurement of an electric current or emitted electromagnetic radiation. For example, the rate of energy inputted into the system can provide a measure of the lnminpcr-pnt species.
Suitable measurements include, for example, mea~ul~ Ls of W096l06946 2 1 98489 . ~ o ~
electric current when the lnminpcrpnt species is generated electrorhPm;c~lly, the rate of reductant or oxidant utilization when the lllm;npccpnt species i5 generated rhPm;r~lly or the absorption of ele~L-~ ~tic energy in S photolnminpcrpnt techniques. In addition, of course, the lllminPcrPmt species can be detected by measuring the emitted ele~Lr~ ~ Lic radiation. All of these measu., L~ can be made either as continuous, rate-based mea~u., L~, or as cumulative methods which ~ te the signal over a long period of time. An example of rate-based mea~uL~ Ls would be by using photomultiplier tubes, photodiodes or phototransistors to produce electric currents proportional in magnitude to the incident light inten_ity. r lpc of cumulative methods are the integration of rate-based data, and the use of photographic film to provide cumulative data directly.
All of these lllminperpnre-based methods entail repeated lnminPccPnre by the ruthenium-containing _ '.
The repetitive nature of the detectable event distinguishes these labels from radioactive isotopes or bound rhPmilnminPcrpnt molecules such as luminol. The latter labels produce a detectable event only once per molecule (or atom) of label, thereby limitiny their detectability.
The following examples illustrate various aspects of the invention but are in no way intended to limit the scope thereof. Analysis was performed in a Plexiglas cell ~PcirnpA
for ECL and electrochemical studies using the film of the wos6/u6s46 PCT~S9~H0630 2~ 98489 present invention, a gold surface supported on the silicon wafer containing r 'allic sites, e.g., Al. A s~uL~ted calomel reference electrode (SCE) and a platinum wire counter electrode were used for all nea~uL~ L8. The electrorhpm;
mea~uL~ Ls coupled with ECL experiments were carried out with a Nodel 175 universal ~LOU~L ~ a Model 173 potentiostat (Princeton Applied r ~ , PAR, Princeton, NJ), and an Ominigraphic 2000 X-Y recorder (Houston In~LL~ n~c, Houston, TX). The ECL emission was detected by a Model C123 single-photu.l cuurlLiny system (Hamamatsu Corp., Bridgewater, NJ) utilizing a Hamamatsu R928P PMT, cooled to -20~C in a Model TE 308 TSRF cooler controller (Products for Research Inc., Danvers, MA). The meter output was fed into the y-axis of the x-y 1~CULd~L, and the signal from the potentiostat was fed into the x-axis to afford ECL intensity versus bias potential display. Solution analysis by ECL was carried out uith a QPCR analyzer (Perkin-Elmer, Norwalk, CT).
A MRC (Materials Research Corporation, Orangeburg, NY) Model 8620 sputtering system at 10-2 torr, with an RF power of 150 W and RF peak to peak voltage of 1.8 KV, was used to sputter gold (99.999~) on silicon wafers.
Polydeoxyadenylic acid (Poly(dA)), polythymidylic acid (poly(dT), polydeoxycytidylic acid (poly(dC)) and calf thymus (CT) ss-DNA and ds-DNA were obtained from Sigma ch~ l Co. (St. Louis, MO) and were used without additional purification. The ss-DNA samples, ~-1 DNA
(5'GAAAATGTGCTGACCGGACATGAA~ATGAG3'), (Seq. ID. No. 1) ~ DNA
W096/06946 2 1 9 8 4 8 9 PCT~595110630 tagged with Ru(bpy)32+(5'Ru(bpy)32+-GAAAATGTGCT~-~CCGc-~c~GAAAATGAG3~), (Seg. ID. No. 2) and a comp]~ ~L~ strand ~-lc DNA
(5'CTCATTTTCA~lCCG~cAGCACATTTTC3'), were obtained from Perkin-Elmer and diluted with a 5 mM tris buffer containing 50 m~ NaCl(p~ 7). Synthesis of ss-DNA can be carried out on a DNA synthesizer (e.g. Applied Biosystems, Model 381A). See also L.J. NcBride and M.H. Cruthers, Tetrahedron Letters, 24, 245 (1983).
The reagents used in the following examples include trichloroethylene (99.6%), 2-propanol (99.9~), tripropylamine (TPrA) (98%), Ru(bpy)3Cl2.6H2O, Ru(phen)3Cl2O, ethyl alcohol (200 proof), Al(NO3)3.9H2O, KICr20~, NaHzPO~ and tris ~hydL uxy ' hyl ) 15 ~mir ~'-n~ and were used as received without purification.
Ri ~I.Iln~.l.. i o acid H2o3p(cH2)4po3H2 (C~BPA)~ and 4 mercaptobutylph~,lh i~ acid (MBPA~ were synthesized in a~L~ e with the techniques taught by Mallouk et al, J.A.C.S., 115, 11855 (1993). The assay buffer for ECL
experiments contained 0.13 M TPrA and 0.19 M phosphate buffer, ple~aled by dissolving TPrA into a NaH2PO~ 601ution and adjusting the pH to 7 with 1 M NaOH.
Deinni~od water from a Millipore Milli-Q (18 Mn-cm) system was used to prepare all aqueous solutions and to rinse the electrode surface.
TEN 8_mple PreparAtion.
TEM samples were prepared by coating Au on a Formvar film on a #400 Cu grid with a vacum evaporator (Edwards 306), ~ W096/06946 2 1 9 8 4 8 9 ~ oo~
fabricating the All(C4BP) film on Au following the procedure described above and then ; h;l;7ing DNA on the Al2(C4BP) film. A trAn~ si~ electron microscope tJE0L lOOCX) at 80 KV was used to image the Au substrate, the Al2(C4BP) film and the i -'-ili7e~ DNA.
W096/06946 2 1 9 8 4 8 ~ PCT~S9~/10630 r~m~le 1 Flectro~eneratea rhp~ eqc~nt Detection of sd do-3NA l-q;nrt A RUthenium L~bel.
Calf thymus ds-DNA was ; -hi 1; zed on the surface of an All(C~BP) film by immersing the film in a solution of DNA
(1.9 m~ in nucleotide phosphate, NP) for 4 h (Figs. 1 and 2).
The film was then rinsed three times with 4-mL portions of ~r;nn;7n5 water and then immersed ;or 4 h in either an agueous 0.56 m~ Ru(phen)3Cl2 solution or a 0.12 mM Ru(phen)3(Cl0~)2 solution in MeCN. Ru(phen)32+ associates with ds-DNA and can be detected through its ele~Lluy~neLaLed chemilllm;n~6c~nre (ECL). Alternatively, the film could be soaked in a mixed ds-DNA (1.9 mM NP) and Ru(phen)3Cl2 (0.12mM) solution for 4 h to produce the ada~LBed layer.
ECL was produced by scanning the potential of the electrode following film formation, DNA adsorption, and Ru(phen)33+ association, from 0 to 1.6 V vs. a saturated calomel electrode (SCE) while it was ; aed in a solution of 0.19 M phosphate buffer (pH 7) containing 0.13 M tri-n-propylamine (TPrA). Typical ECL transients, detected with a single-photon-counting apparatus, are shown in Figures 3A-3C.
Emission arises from the energetic electron-transfer reaction between ele.LLogenerated Ru(phen)33+ and an interm-ediate in the oxidation of TPrA:
DNA-Ru(phen)32+ (ads) - e - DNA-Ru(phen)33+ [1]
CH3CH2CX2-N-Pr2 - e - CHlCH~CHINPrl+ [2]
CH3CHICH.NPr2+ - CH3CH,CHNPr2 + H+ [3]
~ W096/06946 2 ~ 984 89 r~ r DNA-Ru(phen)33+ + CH3CH2CHNPrllDNA-Ru(phen)32++r~,r~,r~NPr2+ [4]
DNA-Ru(phen)32+ ~ DNA-Ru(phen)32+ + hu [5]
The Pm;~ion intensity decreased on a second scan and none was seen on a third, suggesting 1058 of Ru(phen)33+ from the film and diffusion into the bulk solution.
In a control experiment, an electrode with a film of Al2(C4BP) that had not been treated with DNA was soaked for 4 h in either an aqueous 0.56 mN Ru(phen)32+ or a 0.12 mM
Ru(phen)32+ in NeCN solution and then rinsed with NeCN and water. This electrode showed no ECL emission upon potential sweep in the same TPrA solution described above, d~ LL~ting the adsorption of Ru(phen)32+ on the Al2(C4BP) film does not occur. The ability to generate Ru(phen)33+ electrorh~mirAlly in this ECL experiment ~ L~tes that the Al2(C4BP) film and the DNA layer do not prevent het lugeneuus electron-transfer reactions.
To obtain further evidence for Al2(C~BP) film formation and DNA immobilization, experiments were undcrtaken with a gold-coated quartz crystal and a quartz crystal micrnh~l~nre (QCX). The gold was repeatedly treated with hot chromic acid and then rinsed with water and EtOH until the surface was hydrophilic, as indicated by contact angle mea~ur~ L~. The crystal frequency was then measured in air during different stages of Al~(C4BP) film formation, after DNA
adsorption, and after Ru(phen)32+ association. The film was rinsed after each step with deionized water and dried in a W096/06946 2 t ~ 8 4 8 ~ F~l/u~ ~10630 stream of N2 before measurement of the frequency. Results are shown in Table 1.
~ W096/06946 2 1 9 8 4 8 9 PCT~S95110630 T~bln l. F~ ~c~ and Ma~4s Changes on a QCH Plato for Fil_ Growth. DNA I_mobiliz~tion, ~nd Ru~Phen)3l+ Bindinq.
After immersion in: ' ~f~ (Hz) ~m (ns) 109rh (mol/cm NBPA~ -70 137 Al(NO3)3' -27 53 l' C4BPAd -145 283 2 Al(NO3)1' -25 49 l~
ds-DNA' -24 47 0.3i Ru(~hen)~2+~ -13 35 0.l 10 a Immersions were sequential in order given from top to botto_;
bare surface was gold; bare crystal freguency, 6.011329 MHz.
0.5 mM M~3PA in EtOH for - 24 h.
' 5 mM ag. Al(NO3)3 for - 4 h.
d 5 mM ph~1h--~ic acid for 4 h.
' Calf thymus DNA (3.8 mM NP) for 4 h.
f 0.24 mM Ru(phen)32+ in MeCN for 4 h.
The variAtion and drift of the signal over the series of mea~u~ L~ was about + l Hz.
h Ac51lming ro--ghnPcs factor for gold of 2 (total surface area, 0.6 cm2). For comparison, for a close-packed monolayer of MBPA, r = o . 6 x 109 mol/cm2.
i Acc11ming an Al(H2O)33+ until adsorbed.
i Cur~ 7;nq to moles of nucleotide phosphate (NP) per cm2.
- Clearly, the crystal fre~uency decreases as the Al2(C4i3P) film forms and DNA and Ru(phen)32+ are adsorbed on the surface, showing an increase of mass on the crystal during the different stages. The mass change, ~m, can be related to the W096/06946 ~ 2 1 984 8~ P~ oo~o ~
frequency change, ~f, by the Sauerbrey equation:
Qm = -(A ~ /2Fo2)~f where Fo i5 the fnn~ 1 fL~uu~ y of the llnloA~Pd crystal (6 MHz), A is the electrode area (0.159 cm2), pq is the density~
of quartz (2.65 g/cm3) and ~q is the shear modulus of quartz (2.95 x 101l dyne/cm2). With these constants, ~m (ng) = -1.95 ~f (Hz) The mass changes calculated in this way are also given in Table 1. These can be cu-lv~LL~d to approximate surface o ~u~ .LLaLions, r, ACcllming a Luu~l.ne_s factor of 2 (i.e., total surface area of both sides of the quartz crystal of 0.6 cm2). ECL could also be detected from both gold surfaces of the ~uartz crystal, when used as a ~ub~LL~te for Al2(C4BP) film formation, DNA adsorption and ~u(phen)3l+ association and then ~5 scanned in the TPrA solution.
The electrode surface can be designed with immobilized DNA without adsorbing a detector molecule, Ru(phen)32~. A ds-DNA on the surface can be detected by eleu ~ uu~n~L~ted ~hpm;lllm;npcrpn~e of associated Ru(phen)3t~.
Single-stranded DNA can also be i ~;1;7ed on the Alt(C4BP) film surface and then hybridized with complementary DNA in solution with detection of the ds-DNA produced by ECL.
~ W096106946 2 1 9 8 4 8 9 PCT~S95/10630 F!v~mnle 2 8i~qlc ~L~ n'~ i7ation and hvbridiz~tion.
~-1 tagged ss-DNA ti.e., labeled with Ru(bpy)32+) was ; h;li70~ on an Alnm;nnm phosphate film of the present invention by immersing the film in the DNA solution tFig. 4A).
The amount of ; -hili7sd DNA-Ru(bpy)32+ on the surface was tprmino~4~ by ECL resulting from the oxidation of Ru(bpy)l2+
and TPrA in a solution.
Untagged ~-lc ss-DNA was ; i1;70~ on an Alllmin~lm phosphate of the present invention. The ~-lc ss-DNA
containing film was incubated in a complementary strand ~-1 tagged ss-DNA solution at 60OC for 5 min and then cooled to room +E , ~LULC gradually; during this cooling the ss-DNA
hybridized with the compl LaLy strand DNA (Fig. 4B). The hybridized DNA-Ru(bpy)32+ on the film was detected by ECL a8 described above.
Poly(dA) was immobilized on an Alllminllm phosphate film of the present invention by soaking the film in a poly(dA) solution. After the ; ~il;7ation, poly(dT) was hybridized with poly(dA) to produce poly(dA)-poly(dT) ds-DNA
on the surface by incubating the film in a poly(dT) solution at 70~C for 5 min and then cooled to the room temperature gradually (Fig. 4C). To intercalate Ru(phen)32+ into the ds-DNA (poly(dA)-poly(dT)), the Al2(C4BP)/poly(dA)-poly(dT) film was treated with a Ru(phen)3Z+ solution. The hybridized poly(dA)-poly(dT)-Ru(phen)32+ on the surface was detommined by ECL based on the oxidation of Ru(phen)32~ and TPrA in the W096/06946 2 t 9 8 ~ 8 9 r~ o solution.
ExamPle 3 ~E ~ili7~tion ana Detection of 30 bp ss-DNA.
- hi 1~ ~ed on A~ m PhosPh~te Film.
The aluminum phosphate film, prepared as described above was immersed in a 1.38 ~M solution of ~-1 30 bp ss-DNA
~tagged with Ru(bpy)32+) for -2 h. This was employed as a working electrode for an ECL experiment in a 0.19 ~ phosphate buffer (pH 7) containing 0.13 M TPrA. Cyclic vol+, , a~
and t~ cion transients were obtained by scanning the potential of the electrode from O to 1.6 V vs, a saturated calomel electrode (SCE). A L~r ese--Lative voll a~ and t~ -;nn detected with a single-photon-counting system are shown in Figures 5A-5C. The broad oxidation was at - 1.2 V
and a small reductlon wave at -0.35 V observed in the voli a~ (Fig. 5A) arise from the oxidation of TPrA, Ru(bpy)3Z+ and the Au substrate and the reduction of the oxide of Au. The ECL P~iccion from the Al2(C~BP)/~-1 ss-DNA-Ru(bpy)3~+ electrode (Fig. 5B) tl Lr aLes that the ~-1 30 bp ss-DNA-Ru(bpy)3l+ was immobilized on the film. The decay of the light intensity (I) as a function of time (t) was also investigated with the single-photon-counting system. The light intensity t1PtPt'tPtl by the photomultiplier tube, when the potential was stepped from O to 1.50 V, decreased with time (Fig. 6). The decay o~ intensity with time suggests desorption of ss-DNA-Ru(bpy)32+ from the electrode surface or ~ W096/06946 2 1 9 8 4 8 9 PCT~S95/10630 irreversible de _sition of the emitter. Emission results from the energetic electron-transfer reaction between electrogenerated Ru(bpy)33+ and an in+~ te in the oxidation of TPrA:
DNA-Ru(bpy)32+ (ads) I DNA-Ru(bpy)33+ + e t6 CH3CH2CH2NPr2 - e - CH3CH2CH2NPr2 t7]
r~,r~ Npr2+ _ r~Jr~lr~Npr2 + H+ [8]
DNA-Ru(bpy)33++CH3CH2CHNPr2 -- DNA-Ru(bpy)32+ +CT~,r~t'TTNPr2+ [9]
DNA-Ru(bpy)32+ - DNA-Ru(bpy)32+ + hu [10 No ECL Pmi~inn was observed from a film prepared by immersing it in solu+inn of llnllhPlpd ss-DNA: 1.38 ~ A-1 30 bp ss-DNA
or a 0.37 ~N A-lc 30 bp ss-DNA snlntinn (Fig. SC).
A control experiment with the aluminum phosphate film i ped in a 304 n~ Ru(bpy)32+ solution for - 4 h shows a negligible ECL signal, indicating that Ru(bpy)32+ does not adsorb on the film and oxidize.
~-mDle 4 Detectio~ of 30 bD ss-DNA Hvbridized to Com~l D ~ 85-DNA Ta~ed ~Lt~ A Ruthenium Label.
After the ; 'il;7ation of nnl~hPl~d A-lc 30 bp ss-DNA on the Al2(C4BP) film, the film was immersed in a 1.38 ~M
compl L~LY strand ss-DNA (tagged with Ru(bpy)32+) (A-l 30 bp ss-DNA-Ru(bpy)32+) solution. The film in the solution was gradually heated to 60~C in water bath, incubated at 60DC for W096/06946 2 1 9 8 4 8 ~ PCT~S9~10630 5 min. and then slowly cooled to room-t~ e~LuLes, during which the ~-1 ss-DNA-Ru(bpy~ 32+ was hybridized with the compl y strand ~-lc ss-DNA on the surface as ill~L,~Led in Fig. 4B. The film with hybridized DNA was employed as the working electrode in an ECL cell as described above. The ECL
emission from the film was observed as shown in Fig. 7A.
However, when ~-1 ss-DNA, rather than ~-lc ss-DNA, was i hili7n~ on the film and followed by the same hybridization ~L u~eduL ~ as described above by incubating the film in the ~-1 ss-DNA-Ru(bpy)32+ solution, heating to 60~C, and then cooliny to roon-temperature, no obvious ECL emission was evident as shown in Fig. 7B. This d -LL~tes that the ECL nmiCR;~n in Fig. 7A arises from the hybridization of ; - ili 7r~r1 ~-DNA with ~-1 ss-DNA-Ru(bpy)32+ in the solution. The nmiqcirn again results from the oxidation of Ru(bpy)32+-tagged hybridized ss-DNA and TPrA in the solution as described by Eqs. [6] - [10]. In this experiment the Al2(C,BP) film should be incubAted in the A-l ss-DNA solution for a sufficiently long time (at least 4 h) to cover all possible Al (III) binding sites on the film with the ~-1 ss-DNA before the Al2(C4BP)/~-1 ss-DNA electrode is exposed to the h-l ss-DNA-Ru(bpy)32+. It is preferred to cover all Al3+ adsorption sites to avoid r~micc;o~ from the film/~-1 ss-DNA/~-1 ss-DNA-Ru(bpy)32+ arising from some immobilization of ss-DNA-Ru(bpy~ 32+ .
W096/069~6 P~llu~ O
2~ 98489 FYAmnle 5 Detection of ~olv(dA) RYbridized to ~olY~dT) l~ein~ a Ruth~nillm T.~h~l .
The Alllm;n--m pl~n,lll Ate film, pL~a~d as described S above, was immersed in a 21 ~M poly(dA) solution for -4 h, then in a 0.24 mM Ru(phen) 3Z+ solution for -4 h. When the Al2(C~BP)/poly(dA)/Ru(phen)32+ film was employed as a working electrode in a solution of 0.19 M phosphate buffer (pH 7) containing 0.13 M TPrA, no ECL emission was observed (Fig.
8A). This is consistent with the lack of association of Ru(phen)32+ with ss-DNA. However, a film of poly(dA) hybridized with only poly(dT) to form ds-DNA did produce ECL.
This film, formed by immersicn in the poly(dA)solution, was incubated in a 38 ~M poly(dT) solution (gradually heated to 70~C in a water bath, incubated at 70~C for S min and then slowly cooled to room temperature). It was then treated with a 0.24 mM Ru(phen~32+ aqueous solution and used as a working electrode in the TPrA-containing phosphate buffer for ECL
investigation. ECL emission was observed as shown in a representative Pmiqeion transient (Fig. 8B), ~ ~L~ting that poly(dT) in the solution hybridized with the poly(dA) on the surface to produce a ds-DNA [poly(dA)-poly(dT] and Ru(phen) 32+ intercalated with the ds-DNA as shown in Fig. 4c.
These examples show that Ru(phen) 32+ only intercalates with ds-DNA tpoly(dA)-poly(dT], but not with ss-DNA ~poly(dA)). Tntercalation of Ru(phen)32+ with ss-DNA and ds-DNA, ECL emission was measured from a 43 nM Ru(phen)32+
W096106946 PCT~S95/10630 2 1 ~848~ --solution, a 43 nM Ru(phen)32~ containing 57 ~M nucleotide phosphate ([NP]) of calf thymus ss-DNA and a 43 nM Ru(phen)37' containing 33 ~M [NP] of calf thymus ds-DNA with a ~PCR
analyzer. Results are given in Table 2, ~ W096l06946 2 1 9 ~ 4 8 ~ PCT~S9s/10630 T~ble 2 ECL Or 801utions o~ 43 nN Ru(phen~32+
~ith ss-DNA ~nd d~-DNA
Solution I II- IIID
Ru(phen) 32+ alone 65 -DNA+Ru(phen) 32+ ds-DNA+Ru(phen) 32+
Conc. NP(~M) ~ 57 33 ECL(cts/s) 10068_23 10052_12 7779+268 ~ Ratio of moles of nucleotide phosphate (NP) of ss-DNA to Ru(phen) 32+ = 1 . 3 x 10~
b Ratio of moles of nucleotide phosphate tNP) of ds-DNA to Ru(phen) 32+ = 7.7 x 103 indicating that the ECL emission decreased after ds-DNA was added to the Ru(phen)32+ solution. A negligible change, however, in the ECL signal was shown when ss-DNA was added, further ~ LLGting that Ru~phen)32+ only intercalates with ds-DNA, but not with ss-DNA.
A control experiment in which a film, after the immobilization of poly(dA), was incubated in a 21 ~M poly(dA) or a 46 ~ poly(dC) solution instead of poly(dT) (70OC for 5 min then cooled to room-t G~ULe) and then treated with Ru(phen)32+ for -2 h ~Luduce5 no ECL P~iqSin~. This indicates that the Al2(C~BP)/poly(dA) electrode can distinguish a complementary strand of poly(dT) DNA from non-complementary ones.
W096/06946 21 984 89 ~ r ~Q
~Y~le 6 TE~ Im~ges of the film and calf thymu5 ds-DNA.
Samples, prepared by coating Au on a Formvar film on a #400 Cu grid with a vzcuum evaporator, fabricating the ~ m;ml~ phosphate film on the Au as described above, and then ; 'il;7ing DNA on the A12(C,BP) film by immersing the film in a 1.65 mM ~NP] of calf thymus ds-DNA for 4 h, were imaged with a tr~nC~;cc;~n electron mi~L~s~u~e tTEM) at 80 RV. As shown in Figs. 9A-9C the featureless Au substrate (Fig. 9A) shows formation of crystalline islands of Al~(C4BP) film (Fig. 9B) and clumps of DNA (Fig. 9C). The film (Fig. lOA) was then treated with either 1.6~ mM [NP] calf thymus ds-DNA (Fig. lOB) or an identical solution of ds-DNA that had been subjected to sonication for 6 h (Fig. lOC). The results indicate that smaller clumps of DNA are adsorbed on the film after sonication.
A further ;- ~ of the invention i5 shown in Figs. llA and llB. A sensor surface having a multilayer film with bonding groups is provided with a complete set o~
olign~rleotide probes using similar techniques described above for adsorbing ds-DNA and for SS-DNA. See Fig. llA. The sensor surface of Fig. llA is contacted with DNA to be sequenced. The above ~;crlocP~ ECL yLuceduL~s are then used to recognize zones with a complementary sequence. (Fiq. llB).
When chips are used, different types of ss-DNA that make up a test sequence are attached to the surface of a chip to make an array. The chip array is then exposed to the WO 96/06946 2 1 9 8 4 8 q . ~ 10630 sa~ple solution to be sequenced, with formation of ds-DNA at the appropriate location being recognized by the ECL approach.
Although the invention has been described in conjunction with the specific ~ ~nts, it is evident that many alternatives and variations will be apparent to those skilled in the art in light of the foregoing description.
Accordingly, the invention is intpn~p~ to embrace all of the alternatives and variations that fall within the spirit and scope of the AprPn~P~ claims.
WO 96/06946 2 ~ 9 8 4 8 9 . ~ 'Q
~yU~N~ LISTING
(l) GEh-ERAL INFORMATION:
(i) APPLICANTS: Allen J. Lard and Xia-Hong Hu ~ii) TITLE OF THE INVENTION: 3IOSENSOR FOR AND
METHOD OF ELEcTRor~RNRR~TRn cHE~TTlnMTNRqcENT
D~ 1UN aE NUCLEIC ACID ADSORDED TO A SOLID
SURFACE
(iii) NUM8ER OF 9~yu~Nu~S: 3 (iv) CU~i~UN~NU~ ADDRESS:
(A) ~ : Morgan i~ Finnegan (B) T'.EET: 1299 Pennsylvania Ave., N.W., .u.te 960 (C) ~I'Y: ~ashington (D) ~T.TE: Distrlct of rOl ~'A
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(i) SEQUENC. r~R~r~RRT.CTICS:
(A) LE-GTH: 30 (~) TY E: nucleic acid (C) S~ RNnRnNRqS: single ~D) TO OLOGY: linear ~ W096/06946 2 1 9 8 4 8 9 PCT~S95/10630 (ii) MOLECULE TYPE:
(iii) ~Y~Ol~hllCAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: L~3DA
(vi) tlRTr7TN~T SOUR OE :
A) ~t~'TT'qM
'3~ S~ A.N
f, 1 J-.lJJ.L ISOLATE:
D~r:.OP.~NTAL STAGE
' H~'~:,OTY._:
,~, T~.-'UE ~'PE:
C~, C ,, TY:'.':
:: C.,~ LI~.:
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GAAAATGTGC TGACCGGACA TGAA~ATGAG 30 ~2) INFORMATION FOR SEQ ID NO: 2:
W096/06946 21 q 8 4 ~ PCT~S95/10630 (i, S--Q7JENCE C ~RACTERISTICS:
(A LENGT~: 30 (B TY.~E:
(C s~'~NnRnNR.~.~: qingle (D TOPOLOGY: linear (ii) MnT,RrTTT.R TYPE:
(iii) ~Y~ CAL: NO
~iv) ANTI-SENSE: N/A
(v) FRAGMENT TYPE: LAMBDA
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CTCATTTTCA l~l~ l~A GCACATTTTC 30
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W096/06946 21 q 8 4 ~ PCT~S95/10630 (i, S--Q7JENCE C ~RACTERISTICS:
(A LENGT~: 30 (B TY.~E:
(C s~'~NnRnNR.~.~: qingle (D TOPOLOGY: linear (ii) MnT,RrTTT.R TYPE:
(iii) ~Y~ CAL: NO
~iv) ANTI-SENSE: N/A
(v) FRAGMENT TYPE: LAMBDA
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CTCATTTTCA l~l~ l~A GCACATTTTC 30
Claims (20)
1. A method of detecting nucleic acids comprising:
a. forming a sensor surface bearing a film having metallic centers;
b. adsorbing at least one nucleic acid onto to said metal centers;
c. reacting nucleic acid adsorbed to said film with a luminescent metal label; and d. detecting the nucleic acid metal label chelates formed in step (c) via electrogenerated chemiluminescence of said chelates.
a. forming a sensor surface bearing a film having metallic centers;
b. adsorbing at least one nucleic acid onto to said metal centers;
c. reacting nucleic acid adsorbed to said film with a luminescent metal label; and d. detecting the nucleic acid metal label chelates formed in step (c) via electrogenerated chemiluminescence of said chelates.
2. The method of claim 1, wherein the nucleic acid of step (b) is double-stranded.
3. The method of claim 1, wherein the nucleic acid of step (b) is single-stranded.
4. The method of claim 3, wherein step (c) further comprises binding the single-stranded nucleic acid adsorbed to the film, to a complementary single-stranded nucleic acid sequence.
5. The method of claim 1, wherein said electrogenerated chemiluminescence arises from a reaction of an electrogenerated metal label in the nucleic acid -metal label chelate and a suitable coreactant.
6. The method of claim 5, wherein said eletrogenerated metal label is produced via emission from the sensor which is an electrode.
7. A method of detecting nucleic acids comprising:
a. forming an electrode by adsorbing at least one nucleic acid onto said electrode;
b. reacting nucleic acid adsorbed to said electrode with a luminescent label; and c. detecting the nucleic acid label formed in step (b) via electrogenerated chemiluminescence.
a. forming an electrode by adsorbing at least one nucleic acid onto said electrode;
b. reacting nucleic acid adsorbed to said electrode with a luminescent label; and c. detecting the nucleic acid label formed in step (b) via electrogenerated chemiluminescence.
8. The method of claim 7, wherein the nucleic acid of step (b) is double-stranded.
9. The method of claim 7, wherein the nucleic acid of step (b) is single-stranded.
10. The method of claim 8, wherein step (c) further comprises binding the single-stranded nucleic acid adsorbed to the electrode, to a complementary single-stranded nucleic acid sequence.
11. The method of claim 7, wherein said electrogenerated chemiluminescence arises from a reaction of an eletrogenerated metal label in the nucleic acid - metal label chelate and a suitable coreactant.
12. The method of claim 11, wherein said eletrogenerated metal label is produced via emission from the electrode.
13. An aluminum alkanebisphosphonate biosensor comprising:
a substrate and a first metallic layer; and an aluminum alkanebisphosphonate layer forming (A1) metallic centers.
a substrate and a first metallic layer; and an aluminum alkanebisphosphonate layer forming (A1) metallic centers.
14. A biosensor according to claim 13, wherein said biosensor further includes single-strand DNA immobilized to said aluminum centers.
15. A biosensor according to claim 14, wherein said DNA
is labelled with and an osmium or ruthenium moiety.
is labelled with and an osmium or ruthenium moiety.
16. A biosensor according to claim 13, wherein said biosensor further includes double-strand DNA immobilized to said aluminum centers.
17. A biosensor according to claim 16, wherein said DNA
is labelled with an osmium or ruthenium moiety.
is labelled with an osmium or ruthenium moiety.
18. A method of preparing a biosensor for nucleic acid, comprising:
a) treating a silicon wafer to form a chromium layer and juxtaposed gold layer;
b) contacting said layered wafer with an anchoring agent; and c) immersing the product of step b) alternately in Al(NO3)3, bisphosphonic (H2O3P(CH2)4PO3H2) and Al(NO3)3 aqueous solutions.
a) treating a silicon wafer to form a chromium layer and juxtaposed gold layer;
b) contacting said layered wafer with an anchoring agent; and c) immersing the product of step b) alternately in Al(NO3)3, bisphosphonic (H2O3P(CH2)4PO3H2) and Al(NO3)3 aqueous solutions.
19. A biosensor comprising:
an electrode substrate, adsorbed ss-DNA or ds-DNA
immobilized on said electrode, and a detectable label on said ss-DNA or ds-DNA.
an electrode substrate, adsorbed ss-DNA or ds-DNA
immobilized on said electrode, and a detectable label on said ss-DNA or ds-DNA.
20. A biosensor according to claim 19, wherein said DNA
is labelled with an osmium or ruthenium moiety or a luminescent label.
is labelled with an osmium or ruthenium moiety or a luminescent label.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US29663094A | 1994-08-26 | 1994-08-26 | |
| US08/296,630 | 1994-08-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2198489A1 true CA2198489A1 (en) | 1996-03-07 |
Family
ID=23142843
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002198489A Abandoned CA2198489A1 (en) | 1994-08-26 | 1995-08-25 | Biosensor for and method of electrogenerated chemiluminescent detection of nucleic acid adsorbed to a solid surface |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0777741A4 (en) |
| JP (1) | JPH10509025A (en) |
| AU (1) | AU703344B2 (en) |
| CA (1) | CA2198489A1 (en) |
| WO (1) | WO1996006946A1 (en) |
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| US6207369B1 (en) | 1995-03-10 | 2001-03-27 | Meso Scale Technologies, Llc | Multi-array, multi-specific electrochemiluminescence testing |
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| US7138121B2 (en) | 2003-01-23 | 2006-11-21 | Spangler Brenda D | Biosensors utilizing dendrimer-immobilized ligands and there use thereof |
| US7741033B2 (en) * | 2003-05-13 | 2010-06-22 | Trustees Of Boston College | Electrocatalytic nucleic acid hybridization detection |
| ATE445715T1 (en) | 2003-05-13 | 2009-10-15 | Trustees Boston College | ELECTROCATALYTIC NUCLEIC ACID HYBRIDIZATION DETECTION |
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| CN110234995A (en) | 2017-02-02 | 2019-09-13 | 豪夫迈·罗氏有限公司 | Immunoassay using at least two pegylated analyte-specific binding reagents |
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| BR112022000172A2 (en) | 2019-07-22 | 2022-02-22 | Hoffmann La Roche | Methods for assessing whether a patient has endometriosis, for selecting a patient for therapy, and for monitoring a patient |
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| CN111272742B (en) * | 2020-03-06 | 2022-11-08 | 安徽大学 | Electrochemiluminescence sensor based on metal organic gel composite material and metal organic framework and preparation and detection methods thereof |
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| WO2022207628A1 (en) | 2021-03-30 | 2022-10-06 | F. Hoffmann-La Roche Ag | Scf as blood biomarker for the non-invasive diagnosis of endometriosis |
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| GB1258580A (en) * | 1967-12-28 | 1971-12-30 | ||
| US5238808A (en) * | 1984-10-31 | 1993-08-24 | Igen, Inc. | Luminescent metal chelate labels and means for detection |
| US5124022A (en) * | 1989-08-23 | 1992-06-23 | Aluminum Company Of America | Electrolytic capacitor and method of making same |
| US5019343A (en) * | 1989-12-15 | 1991-05-28 | W. R. Grace & Co.-Conn. | Control of corrosion in aqueous systems using certain phosphonomethyl amines |
| US5846708A (en) * | 1991-11-19 | 1998-12-08 | Massachusetts Institiute Of Technology | Optical and electrical methods and apparatus for molecule detection |
-
1995
- 1995-08-25 WO PCT/US1995/010630 patent/WO1996006946A1/en not_active Application Discontinuation
- 1995-08-25 JP JP8508822A patent/JPH10509025A/en active Pending
- 1995-08-25 AU AU34103/95A patent/AU703344B2/en not_active Ceased
- 1995-08-25 EP EP95930881A patent/EP0777741A4/en not_active Withdrawn
- 1995-08-25 CA CA002198489A patent/CA2198489A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU703344B2 (en) | 1999-03-25 |
| AU3410395A (en) | 1996-03-22 |
| EP0777741A1 (en) | 1997-06-11 |
| JPH10509025A (en) | 1998-09-08 |
| WO1996006946A1 (en) | 1996-03-07 |
| EP0777741A4 (en) | 1999-01-13 |
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