WO2000065099A1 - Monolayer and electrode for detecting a label-bearing target and method of use thereof - Google Patents
Monolayer and electrode for detecting a label-bearing target and method of use thereof Download PDFInfo
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- WO2000065099A1 WO2000065099A1 PCT/US2000/002976 US0002976W WO0065099A1 WO 2000065099 A1 WO2000065099 A1 WO 2000065099A1 US 0002976 W US0002976 W US 0002976W WO 0065099 A1 WO0065099 A1 WO 0065099A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
Definitions
- This invention relates to modified electrodes for analysis of binding pair interactions and the use of these electrodes, especially in nucleic acid analysis and protein-protein interactions.
- the present invention relates to electrodes for detecting interactions between members of a binding pair, which electrodes have been modified by formation of a non-conductive self-assembled monolayer, and to the method of detecting biomolecules, such as nucleic acids or other targets, including receptors, ligands, antigens or antibodies, utilizing such electrodes.
- indirect detection of hybridization can be accomplished using sandwich assays where the surface-bound hybrid is subsequently hybridized to an additional signal probe that carries one or more fluorescent labels or enzymes that convert a non-fluorescent substrate to a fluorescent one (Spargo, CA. et al., 1993, Molecular and Cellular Probes 7, 395- 404).
- sandwich assays where the surface-bound hybrid is subsequently hybridized to an additional signal probe that carries one or more fluorescent labels or enzymes that convert a non-fluorescent substrate to a fluorescent one
- an additional signal probe that carries one or more fluorescent labels or enzymes that convert a non-fluorescent substrate to a fluorescent one
- Organosilanes may be covalently attached at selected positions of a hydroxylated surface of a substrate, such as silicon dioxide, to form an organosilane monolayer or bilayer film or coating, as set forth in the patent of Chrisey et al. (U.S. Patent No. 5,688,642).
- Organosilanes are used that have at least one reactive site for binding to the hydroxylated surface of the substrate and another reactive site that is incapable of binding either to other organosilane molecules of the coating or to the substrate, but is available for binding to a molecule distinct from these, such as a nucleic acid modified by the addition of a thiol or amino group.
- the DNA apparently remains attached through its thiol end group while contacts between DNA backbones and the surface are prevented by the formation of a mercaptohexanol monolayer.
- the oligomeric nucleic acid probe readily hybridizes to its complementary sequence. (Levicky, R. et al., 1998, J. Amer. Chem. Soc, 120, 9787).
- Other systems that have been designed utilizing direct electron transfer from nucleic acids which have been contacted with an electrode, but do not use mediated electron transfer nor a self-assembled monolayer include those of Hall et al., PCT/GB93/00631.
- carboxylate-functionalized ruthenium bipyridyl complexes may be used together with high-area nanocrystalline titanium dioxide films as one way to obtain surface attachment.
- Another way to accomplish surface attachment to nanocrystalline TiO 2 in film (electrode) or colloidal form, and for subsequent retention of the molecule over a wide pH range is hexaphosphonation of Ru(bpy) 3 2+ (Yan, S.G.et al., 1996, J. Physical Chem., 100, 6867).
- This prior technique does not relate to mediated solution electrochemistry as in the current invention, but rather relates to direct electron transfer, using light as a stimulus instead of a voltage.
- the 1,12 dodecane dicarboxylic acid monolayer is not stable under thermal stress, and, compared to the phosphonate of the invention herein, the carboxylate monolayer does not form reproducibly due to its lower stability.
- self-assembled monolayers had not been described that allowed for straightforward attachment of oligonucleotide probes and the electrochemical detection of immobilized DNA via guanine oxidation.
- the self- assembled monolayers of this invention are thermally stable, oxidation resistant, and are formed rapidly and reproducibly.
- carboxylate is used as the terminal group, nonspecific binding is minimized.
- the preferred phosphonate compounds which are used in the invention were previously unavailable or very difficult to synthesize (with only the C 3 carboxy phosphonate and the C 3 amino phosphonate being known to be commercially available).
- Prior work with other phosphonate compounds has been in solution, for example, to enhance chromatographic separation (Lukes, I. et al., 1994, J. Am. Chem.
- target biomolecules such as nucleic acids or other targets, including receptors, ligands, antigens or antibodies.
- the invention herein is a self-assembled phosphonate monolayer on an electrode, which in the preferred embodiment is a carboxy-alkyl phosphonate on an ITO surface, to which a member of a binding pair is covalently bound.
- the invention herein also includes a method of using the monolayer material to form a self- assembled monolayer on an electrode surface and a method of immobilizing binding pair members on the modified electrode surface.
- the electrode with the self- assembled monolayer in a preferred embodiment is useful for the electrochemical detection of a preselected base in a nucleic acid and for determining the presence of a target nucleic acid in a sample.
- an oligonucleotide probe coupled to the self-assembled monolayer reacts with the target nucleic acid to form a hybridized nucleic acid on the modified electrode surface.
- the hybridized nucleic acid is reacted with a transition metal complex capable of oxidizing a preselected base in the hybridized nucleic acid in an oxidation-reduction reaction, the oxidation-reduction reaction is detected; and the presence or absence of hybridized nucleic acid determined from the detected oxidation-reduction reaction.
- the oxidation-reduction reaction can be detected in accordance with the present invention because following the transfer of electrons from the immobilized binding pair to the transition metal complex, the monolayer permits the transition metal complex to transport the electrons to the surface of the electrode, where they are detected.
- the self-assembled monolayer is non-conductive, serves to immobilize reactants near the electrode surface, and allows the transition metal complex to move freely from the immobilized reactants to the conductive working surface of the electrode to permit electron transfer.
- amplification techniques as are known in the art may be used in conjunction with the invention.
- the invention may also be used to detect other targets (e.g, receptors, ligands, antigens, antibodies, etc.).
- targets e.g, receptors, ligands, antigens, antibodies, etc.
- target protein in a sample may be detected by reacting the target protein with a protein binding substance such as an antibody attached to the self-assembled monolayer of the invention, followed by addition of a second protein-binding substance such as a second antibody that has bound to it a label capable of being oxidized in an oxidation-reduction reaction.
- the label is reacted with a transition metal complex capable of oxidizing the label in an oxidation-reduction reaction. Detection of the oxidation-reduction reaction allows determination of the presence or absence of the target protein.
- One label suitable for use in this invention is an oligonucleotide.
- Figures la and lb are graphs of the amount of guanine-containing oligonucleotide bound, in picomoles, versus assembly time of the self-assembled monolayer of the invention, illustrating the effect of self-assembly time on monolayer formation as a function of the amount of oligonucleotide probe that the monolayer is able to bind.
- Figure 2 graphically illustrates the effect of the concentration of C 12 carboxyphosphonate in the self-assembly solution on monolayer formation, as indicated by the electrochemical response of the guanine-containing oligonucleotide coupled to the self-assembled monolayer. Electrochemical measurements were made using cyclic voltammetry at a scan speed of 20 V/s and a Ru(bpy) 3 2+ concentration of 100 ⁇ M.
- Figure 3 graphically illustrates the stability of the guanine-containing oligonucleotide monolayer made according to the invention using the 12-carbon carboxy-alkyl phosphonate. Electrochemical measurements were made using cyclic voltammetry at a scan speed of 20 V/s and a Ru(bpy) 3 2+ concentration of 100 ⁇ M.
- Figure 4 is a series of cyclic voltammograms illustrating a dose response for oligonucleotide-coupled monolayers that have varying amounts of guanine-containing oligonucleotide attached. Electrochemical measurements were made using cyclic voltammetry at a scan speed of 20 V/s and a Ru(bpy) 3 2+ concentration of 100 ⁇ M. Figure 5 graphically illustrates the dose response shown in Figure 4.
- Electrochemical measurements were made using cyclic voltammetry at a scan speed of 20 N/s and a Ru(bpy) 3 2+ concentration of 100 ⁇ M.
- the present invention provides a self-assembled phosphonate monolayer with a covalently bound member of a binding pair on an electrode and the method of use of these self-assembled monolayers.
- non-conductive "monolayer” includes a single layer covering the conductive working surface of the electrode, preferably comprising alkyl phosphonate self-assembled from solution onto an ITO electrode surface, in what has been termed a "dative” or “coordination” bond between the oxygens on the phosphonate and metal atoms in the electrode (in the preferred embodiment).
- Formation of the monolayer of the invention does not include formation of truly covalent bonds, such as are found between two non-metals, such as C, N and O, which are able to share electrons to form a true covalent bond.
- the invention herein also does not include polymer membranes.
- the self-assembled monolayer of the invention is formed from phosphonate molecules capable of adhering to and modifying an electrode, such as an ITO electrode.
- the molecules used in the invention are multifunctional having at the minimum at least one surface-active functional group that bonds to the ITO surface, preferably phosphonate, and at least one end terminal group, R ] s such as carboxy, amino, hydroxyl or methyl, to which a member of a binding pair is covalently bound.
- these molecules may have an organic spacer R 2 , preferably containing one or more carbon atoms and the associated substituents (generally hydrogen), between the phosphonate group and the R, group.
- the term "member of a binding pair” includes all biomolecules which can bind to one another such as nucleic acids, receptors, ligands, antibodies, antigens and carbohydrates. While the examples herein primarily relate to use of oligonucleotides, one of ordinary skill in the art could use these examples and the disclosure herein for other biomolecules.
- the electrode used in the invention comprises a conductive substrate or a substrate with the outer surface functioning as a conductive working surface.
- the substrate may itself be conductive or it may be nonconductive but have a conductive working surface.
- the electrode can have any shape that is conventional in this art, such as a cylindrical electrode having a conductive working surface on the exterior thereof or a flat sheet having the conductive working surface formed on one side thereof.
- the conductive substrate upon which the monolayer is assembled can be any metal or non-metal material conventionally used, including carbon, such as graphite, glassy carbon, pyrolytic graphite, carbon paste, and carbon fiber; doped and undoped oxides, such as indium-doped tin oxide (ITO), tin oxide, titanium oxide, manganese oxide, and lead oxide; and semiconductor materials, such as Si, Ge, ZnO, CdS, TiO 2 , and GaAs; and the like. It is preferred to use ITO because its properties are relatively well-known, because it is inexpensive, and because it has a high oxidative potential limit in water at neutral pH and a relatively low charging current. The invention will be further described in connection therewith. Metals such as gold having adsorbed thiols or disulfides cannot be used with this invention, because they will oxidize at potentials lower than those needed for guanine oxidation.
- An apparatus for determination of the presence of a label-bearing target may, for example, include a sample container for holding a fluid sample; an electrode, comprising a substrate having a conductive working surface thereon; and a non- conductive self-assembled monolayer on said conductive working surface, said monolayer comprising phosphonate molecules, each of which phosphonate molecules has at the minimum at least one phosphonate group and at least one R, group, where the R ] group is covalently bound to a member of a binding pair, through which monolayer a transition metal complex can freely move from immobilized reactants to the conductive working surface to transfer electrons to the conductive working surface; and a potentiostat in electronic communication with the monolayer.
- the apparatus further comprises a member of a binding pair, such as an oligonucleotide probe attached to the self-assembled monolayer or a protein-binding substance attached to the self-assembled monolayer.
- the method of determining the presence of a target nucleic acid in a sample comprises contacting a monolayer self-assembled on an electrode with an oligonucleotide probe so that the oligonucleotide probe becomes covalently attached to the monolayer; contacting the probe-modified monolayer on the electrode with the nucleic acid solution so that the target nucleic acid and the oligonucleotide probe form a hybridized nucleic acid on the modified electrode; reacting the hybridized nucleic acid with a transition metal complex capable of oxidizing a preselected base in the hybridized nucleic acid in an oxidation-reduction reaction; detecting the oxidation- reduction reaction; and determining the presence or absence of the nucleic acid from the detected oxidation-reduction reaction.
- the oligonucleotide probe may be coupled to the phosphonate before monolayer assembly on the electrode.
- determining the presence of a target protein in a sample comprises contacting a monolayer self-assembled on an electrode with a protein- binding substance so that the binding substance becomes covalently attached to the monolayer according to the invention; contacting the protein-modified monolayer on the electrode with the sample; contacting the modified electrode with a second protein- binding substance that has been modified to contain a label; reacting the monolayer with a transition metal complex capable of oxidizing the label in an oxidation- reduction reaction; detecting the oxidation-reduction reaction; and determining the presence or absence of the target protein from the detected oxidation-reduction reaction.
- One label suitable for use in this invention is an oligonucleotide.
- the protein-binding substance may be coupled to the phosphonate monolayer before monolayer assembly on the electrode.
- Phosphonates As discussed earlier, the molecules forming the self-assembled monolayer of the invention comprise a phosphonate group (-PO 3 H 2 ), which bonds to the ITO electrode surface, and an R, group, which is capable of covalent bonding with a member of a binding pair.
- the phosphonate group may be monophosphonate, diphosphonate, triphosphonate, tetraphosphonate, or polyphosphonate moieties.
- the R, terminal group includes, but is not limited to, carboxyl, acid halide, acid anhydride, hydroxyl, epoxide, aldehyde, ketone, sulfhydryl, nitrile and amino groups.
- the phosphonate group and R are bridged by the organic spacer or linker R 2 , which, when present, may comprise alkyl, alkenyl, alkynyl, and aromatic structures, that may be linear, branched, cyclic or polymeric structures.
- R 2 may be substituted with any number of phosphonate molecules that can bind to the ITO surface.
- the phosphonate source is a carboxy-alkyl phosphonate.
- the phosphonate portion of a carboxy-alkyl phosphonate binds to ITO, with increased stability as the number of carbons in the alkyl portion increases.
- the carboxy moiety gives a negative charge to the monolayer and it is the carboxy group that couples to the member of the binding pair.
- An amino- alkyl phosphonate can be used if a positive monolayer charge is desired and a methyl or hydroxyl phosphonate can be used for a neutral surface charge.
- the carboxy-alkyl phosphonate that is most preferred, based on tests done to date, is the 12-carbon carboxy-alkyl phosphonate (also referred to herein as 11- carboxyundecane phosphonic acid)(see Example 2 for preferred new method of preparation).
- This 12-carbon phosphonate was used primarily in the tests described in the Examples herein. Tests indicate that carboxy-alkyl phosphonates having 2-14 carbons in the alkyl group work to form a self-assembled monolayer with sufficient stability and the characteristics required for use in the invention herein.
- Example 2 generally involves: (1) converting bromoalkyl carboxylic acids to acid chloride intermediates by reaction with oxalyl chloride; followed by (2) converting the acid chloride intermediates into bromoalkyl esters by reaction with an alcohol, such as ethanol, under alkaline conditions so that (3) the bromoalkyl ester intermediates can be converted into carboxy-alkyl phosphonates by reaction with tri ethyl phosphite (or trimethyl phosphite) followed by acid hydrolysis to regenerate the acid.
- an alcohol such as ethanol
- the self-assembled phosphonate monolayer discussed in the examples herein is chemically homogeneous, i.e., consists of only carboxy-alkyl phosphonates.
- the invention includes, however, self-assembled heterogeneous monolayers in which these phosphonates are supplemented with other materials, for example, amino-alkyl phosphonates, hydroxy-alkyl phosphonates, methoxy-alkyl phosphonates, methyl- alkyl phosphonates, thiol-alkyl phosphonates, aldehyde-alkyl phosphonates, trifluoromethyl-alkyl phosphonates and zwitterionic phosphonates of various lengths, that alter the physical and chemical characteristics of the monolayer, for example, overall charge or charge distribution on the monolayer.
- Test samples may be carried out on a test sample containing the target nucleic acid or other target biomolecules such as proteins. Any test sample suspected of containing the target may be used, including, but not limited to, tissue samples such as biopsy samples and biological fluids such as blood, sputum, urine and semen samples, bacterial cultures, soil samples, food samples, cell cultures, etc.
- the target may be of any origin, including animal, plant or microbiological (e.g., viral, prokaryotic, and eukaryotic organisms, including bacterial, protozoal, and fungal, etc.) depending on the particular purpose of the test. Examples include surgical specimens, specimens used for medical diagnostics, specimens used for genetic testing, environmental specimens, cell culture specimens, food specimens, dental specimens and veterinary specimens.
- the sample may be processed or purified prior to carrying out the instant method in accordance with techniques known or apparent to those skilled in the art; and nucleic acids therein may be digested, fragmented, and/or amplified (see below) prior to carrying out the instant method, if so desired. Amplification.
- the processes utilizing an electrode having a self- assembled monolayer according to the present invention involve contacting the target nucleic acid sample to an oligonucleotide probe to produce a hybridized nucleic acid, it may be desirable for certain applications using the invention to amplify the nucleic acid prior to contacting with the oligonucleotide probe.
- Amplification of a selected, or target, nucleic acid sequence may be carried out by any suitable means, such as those disclosed and discussed in the co-pending applications (SN 09/179,665 and SN 08/950,503).
- an electrode of the invention herein on which the self-assembled monolayer has been formed and methods of utilizing this electrode enable detection of hybridized nucleic acid.
- a target nucleic acid is contacted with an oligonucleotide probe bound to a self-assembled monolayer to form a hybridized nucleic acid.
- the oligonucleotide probes which are useful in the methods of the present invention can be any probe comprised of between about 4 or 6 bases up to about 80 or 100 bases or more, more preferably between about 8 and about 30 bases. Oligonucleotide probes may be prepared having any of a wide variety of base sequences according to techniques which are well known in the art.
- Suitable bases for preparing the oligonucleotide probe may be selected from naturally occurring nucleotide bases such as adenine, cytosine, guanine, uracil, and thymine; and non- naturally occurring or "synthetic" nucleotide bases such as 8-oxo-guanine, 6- mercaptoguanine, 4-acetylcytidine, 5-(carboxyhydroxyethyl)uridine, 2'-O- methylcytidine, 5-carboxymethylamino-methyl-2-thiouridine, 5- carboxymethylaminomethyluridine, dihydrouridine, 2'-O-methylpseudouridine, ⁇ ,D- galactosylqueosine, 2'-O-methylguanosine, inosine, 7-deazaguanine, N6- isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1- methylguanosine
- oligonucleotide backbone may be employed, including DNA, RNA, modified sugars such as carbocycles, and sugars containing 2' substitutions such as fluoro and methoxy.
- the oligonucleotides may be oligonucleotides wherein at least one, or all, of the internucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphonothioates, phosphoromorpholidates, phosphoropiperazidates and phosphoramidates (for example, every other one of the internucleotide bridging phosphate residues may be modified as described).
- the oligonucleotide may be a "peptide nucleic acid" such as described in P. Nielsen et al., 1991, Science 254, 1497-1500.
- the only requirement is that the oligonucleotide probe should possess a sequence at least a portion of which is complementary to a portion of the sequence of the target nucleic acid. It may be desirable in some applications to contact the nucleic acid sample with a number of oligonucleotide probes having different base sequences (e.g., where there are two or more target nucleic acids in the sample, or where a single target nucleic acid is hybridized to two or more oligonucleotide probes in a "sandwich" assay).
- Preselected base After hybridization, the target nucleic acid hybridized to the oligonucleotide probe attached to the monolayer self-assembled on the electrode is reacted with a suitable mediator which is capable of oxidizing a preselected base in an oxidation-reduction reaction.
- the preselected base can be any naturally occurring or synthetic nucleotide base which undergoes oxidation upon reaction with the selected mediator.
- the preselected base exhibits a unique oxidation rate when paired as compared to when the preselected base is unpaired.
- the preselected base should exhibit unique oxidation rates when paired with each of the four naturally occurring bases.
- bases whose 5'-mononucleotides e.g., the 5'-deoxyribonucleotide or 5'-ribonucleotide
- bases whose 5'-mononucleotides e.g., the 5'-deoxyribonucleotide or 5'-ribonucleotide
- rate constants above 10 4 M " 's "1 can be detected using the catalytic reaction.
- Suitable preselected bases include but are not limited to guanine, adenine, 8-oxo-guanine, 8-oxo-adenine, 8-bromo-guanine, xanthine, pseudouridine, 6-mercaptoguanine, 8-mercaptoguanine, 2-thioxanthine, 6- thioxanthine, 6-mercaptopurine, 2-amino-6-carboxymethyl-mercaptopurine, 2- mercaptopurine, 6-methoxypurine, 2-acetylamino-6-hydroxypurine, 6-methylthio-2- hydroxypurine, 2-dimethylamino-6-hydroxypurine, 2-hydroxypurine, 2-aminopurine, 6-amino-2-dimethylallyl-purine, 2-thioadenine, 8-hydroxyadenine, and 8- methoxyadenine.
- the preselected base is selected from the group consisting of guanine, adenine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine, with guanine being the currently preferred naturally occurring preselected base and 8-oxo- guanine or 6-mercaptoguanine the currently preferred synthetic preselected base.
- the mediator that is needed to enable electron transfer may be any molecule such as a cationic, anionic, non-ionic, or zwitterionic molecule which is reactive with the preselected base at a unique oxidation potential to transfer electrons from the nucleic acid to the electrode.
- mediator will be dependent upon the particular preselected base chosen, and will be readily determinable by those skilled in the art.
- Particularly preferred mediators include transition metal complexes which are capable of metal-nucleic acid electron transfer with the preselected base such that the reduced form of the metal complex is regenerated, completing a catalytic cycle.
- Some anionic complexes useful as mediators are Ru(bpy)((SO 3 ) 2 -bpy) 2 2" and Ru(bpy)((CO 2 ) 2 -bpy) 2 2" and some zwitterionic complexes useful as mediators are Ru(bpy) 2 ((SO 3 ) 2 -bpy) and Ru(bpy) 2 ((CO 2 ) 2 -bpy) where (SO 3 ) 2 -bpy 2" is 4,4'-disulfonato-2,2'-bipyridine and (CO ⁇ -bpy 2" is 4,4'-dicarboxy-2,2'-bipyridine.
- Suitable substituted derivatives of the pyridine, bypyridine and phenanthroline groups may also be employed in complexes with any of the foregoing metals.
- Suitable substituted derivatives include but are not limited to 4-aminopyridine, 4-dimethylpyridine, 4-acetylpyridine, 4-nitropyridine, 4,4'-diamino-2,2'-bipyridine, 5,5'-diamino-2,2'-bipyridine, 6,6'-diamino-2,2'- bipyridine, 4,4'-diethylenediamine-2,2'-bipyridine, 5,5'-diethylenediamine-2,2'- bipyridine, 6,6'-diethylenediamine-2,2'-bipyridine, 4,4'-dihydroxyl-2,2'-bipyridine, 5,5'-dihydroxyl-2,2'-bipyridine, 6,6'-dihydroxyl-2,2'-bipyridine, 4,4',
- the mediator may be reacted with the hybridized nucleic acid under conditions sufficient to effect the oxidation-reduction reaction of the mediator with the preselected base.
- the solvent in which the oxidation- reduction reaction takes place may be any suitable solvent for solubilizing nucleic acids, and preferably comprises water. Suitable conditions for permitting the oxidation-reduction reaction to occur will be known to those skilled in the art. Detection of oxidation-reduction reaction.
- the occurrence of the oxidation-reduction reaction may be detected on an electrode having a self-assembled monolayer in accord with the present invention to observe a change in the electronic signal which is indicative of the occurrence of the oxidation-reduction reaction.
- the electrode is placed in contact with the solution of mediator and generally, a reference electrode and an auxiliary electrode are also placed in contact with the solution in conjunction with the working electrode (with most of the current passing through the auxiliary electrode).
- suitable reference electrodes will also be known in the art and include, for example, silver/silver chloride electrodes.
- a suitable auxiliary electrode is a Pt electrode.
- the detection of the electronic signal associated with the oxidation-reduction reaction permits the determination of the presence or absence of target.
- the step of determining the presence or absence of target typically includes ( ) measuring the reaction rate of the oxidation-reduction reaction, (ii) comparing the measured reaction rate to the oxidation-reduction reaction rate of the transition metal complex with or without nucleic acid, and then (Hi) determining whether or not the measured reaction rate is essentially the same as the oxidation-reduction reaction rate of the transition metal complex with or without target.
- the step of measuring the reaction rate may be carried out by any suitable means.
- the relative reaction rate may be determined by comparing the current at the same scan rate, probe concentration, target concentration, mediator, buffer, temperature, and/or electrochemical method.
- the oxidation-reduction reaction rate may be measured according to suitable means known to those skilled in the art.
- the oxidation-reduction reaction rate is measured by measuring the electronic signal associated with the occurrence of the oxidation-reduction reaction.
- the electronic signal associated with the oxidation-reduction reaction may be measured by providing a suitable apparatus in electronic communication with an electrode coated with a self-assembled monolayer as disclosed herein.
- a suitable apparatus is a potentiostat capable of measuring the electronic signal which is generated so as to provide a measurement of the oxidation- reduction reaction rate of the reaction between the hybridized nucleic acid and the mediator.
- the detector has bound to it a label capable of being oxidized in an oxidation-reduction reaction, with one such label being an oligonucleotide containing the preselected base.
- the electronic output may be characteristic of any electrochemical method, including cyclic voltammetry, normal pulse voltammetry, chronoamperometry, chronocoulometry, or square-wave voltammetry, with cyclic voltammetry and chronoamperometry being the currently preferred forms.
- a computer as is known in the art may be used for controlling the use of the electrode and for recording results of such use.
- the method most frequently used to analyze nucleic acids on monolayers self-assembled on ITO electrodes according to the invention is cyclic voltammetry. In cyclic voltammetry, the potential of the electrochemical system is varied linearly from an initial potential (0-800 mV) to a final potential (1300-2000 mV).
- the scan direction is reversed and the same potential range is swept in the opposite direction.
- the potential is varied at a constant scan rate (for example, about 10 mV/s to about 5000 V/s).
- the initial potential is set at 0 mV and the final potential experimentally determined by the scan rate.
- the currently preferred scan rate is 20 V/s with a 1600 mV final potential.
- the current is collected at each potential and the data is plotted as a current versus potential spectra.
- potential step methods such as chronocoulometry or chronoamperometry may be used to analyze nucleic acids on monolayers of the invention.
- the electrochemical system is stepped directly from the initial potential (0 mV-800 mV) to the final potential (1000 mV-1600 mV).
- the electrochemical system is held at the final potential for some specified period of time (50 ⁇ s to 30 s) and the charge is collected as a function of time.
- the potential can be stepped back to the initial potential and the charge can be collected at the initial potential as a function of time.
- the electrochemical system is stepped from an initial potential (0 mV-800 mV) directly to a final potential (1000-1600 mV) for some specified period of time (50 ⁇ s to 30 s) and the current is collected as a function of time.
- the potential can be stepped back to the initial potential, and the current can be collected at the initial potential as a function of time.
- the preferred potential step is 1100 mV with collection time of 500 ms although preferred potential steps and times may vary with different assay parameters. Method of detection.
- Detection of a preselected base on a target nucleic acid using an electrode with a non-conductive self-assembled monolayer comprises (a) contacting the test sample to an oligonucleotide probe, bound to a monolayer according to the invention, that specifically binds to the target nucleic acid to form a hybridized nucleic acid; (b) contacting the hybridized nucleic acid to a transition metal complex that oxidizes the preselected base in an oxidation- reduction reaction; (c) detecting the presence or absence of the oxidation-reduction reaction associated with the hybridized nucleic acid; and (d) determining the presence or absence of the target nucleic acid in the test sample from the detected oxidation- reduction reaction at the preselected base.
- the target nucleic acid contains at least about 10 more of the preselected base than does the oligonucleotide probe, or more preferably at least 50 or 100 more of the preselected base than does the oligonucleotide probe.
- a larger current enhancement is advantageously obtained when the target nucleic acid contains many more of the preselected base than does the oligonucleotide probe.
- the target nucleic acid is preferably longer than the oligonucleotide probe, and at least one of the preselected bases is "overhanging" as described in U.S. Patent No. 5,871,918 and not hybridized to the oligonucleotide probe in the hybridized nucleic acid.
- at least 10, 50, or 100 of the preselected bases are "overhanging" bases, thereby providing substantial amplification of the electrochemical signal detected.
- the oligonucleotide probe sequence is free of the preselected base.
- the strategy of employing alternate bases that are redox inactive may be employed.
- an oligonucleotide probe sequence that does not contain any guanine residues (e.g., only A, T, and C) may be chosen.
- the cyclic voltammogram of Ru(bpy) 3 2+ in the presence of this strand is very similar to that without the oligomer.
- This oligonucleotide probe is then hybridized to a target nucleic acid strand that contains guanines in either the overlapping base-paired regions and/or in overhanging regions if the target nucleic acid is longer than the oligonucleotide probe. Because multiple guanines are detected, the signal is amplified relative to the number of hybrids formed. In a case where a genomic DNA or RNA is the target nucleic acid strand, large numbers of overhanging guanines are encountered, which would give tremendous signal amplification.
- the assay for the preselected base on the target nucleic acid strand involves immobilization of the (preferably redox-silent) oligonucleotide probe strand on the self-assembled monolayer on the electrode surface, which provides a low background signal when scanned in the presence of the mediator.
- the monolayer is then contacted with a solution of the target nucleic acid, which contains the preselected base. If hybridization occurs, the target nucleic acid will now be in close proximity to the electrode, and a current enhancement will be detected in the presence of mediator.
- An alternate base may be used that would substitute for guanine (i.e., a base that, like guanine, has a greater binding affinity for cytosine than do other bases in a nucleic acid duplex) in the oligonucleotide probe strand but would not be oxi ⁇ ized by the mediator under the applicable reaction conditions.
- the preselected base in the target nucleic acid is guanine and the target nucleic acid also contains cytosine (which ordinarily bonds with guanine in the oligonucleotide probe)
- the probe contains an alternate base that bonds to cytosine in the hybridized nucleic acid.
- the alternate base may be inosine which is three orders of magnitude less electrochemically reactive than guanine.
- the reacting step typically comprises reacting the transition metal complex with the nucleic acid under conditions sufficient to effect the selective oxidation of the preselected base without oxidizing the alternate base.
- a method of detecting a target nucleic acid comprises: (a) contacting the target nucleic acid to a complementary oligonucleotide probe that specifically binds to the target nucleic acid to form a hybridized nucleic acid; (b) reacting the hybridized nucleic acid with a transition metal complex capable of oxidizing the preselected base in an oxidation- reduction reaction; (c) detecting the oxidation-reduction reaction; and (d) determining the presence or absence of the hybridized nucleic acid from the detected oxidation- reduction reaction at the preselected base.
- the rate constant for oxidation of the hybridized nucleic acid by the mediator can be determined from the cyclic voltammogram by digital simulation.
- This quantity is then related directly to the quantity of material containing target nucleic acid (e.g., infectious organism in a clinical sample). See, e.g., M. Holodniy et al., 1995, J. Virol. 69, 3510-3516; J. Mellors et al., 1996, Science 272, 1167-1170.
- target nucleic acid e.g., infectious organism in a clinical sample.
- the monolayer self-assembled onto the conductive working surface of an electrode can also be used for detection of biomolecules other than nucleic acids such as proteins using the disclosure of the invention herein along with methods of working with proteins known to those of skill in the art. As with nucleic acids, no enzyme label is required for use of the invention with other biomolecules.
- a method of detecting a target protein in a sample comprises: (a) attaching a protein-binding substance to a monolayer that has been self- assembled onto a conductive working surface; (b) contacting the target protein to the protein-binding substance coupled to the monolayer; (c) contacting the target protein bound to the monolayer to a second protein-binding substance which has bound to it a label capable of being oxidized in an oxidation reduction reaction; (d) reacting the label on the second protein-binding substance bound to the target protein with a transition metal complex capable of oxidizing the label in an oxidation-reduction reaction; (e) detecting the oxidation-reduction reaction; and (f) determining the presence or absence of the target protein from the detected oxidation-reduction reaction.
- Example 1 Reagents and DNA. Inorganic reagents used in these experiments were of analytical grade or higher. The sources of the reagents are as follows: carboxy-alkyl phosphonates made according to Example 2 or by Sigma Chemicals (St. Louis, MO) or Aldrich (Milwaukee, WI); l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and ethanolamine (Sigma or
- Example 2 Preferred Method ofC-12 Phosphonate Preparation.
- certain phosphonic acids are currently commercially available, for example, amino propyl phosphonic acid and 2-carboxy ethyl phosphonic acid (Sigma or Aldrich), it is preferred to utilize a higher carbon phosphonic acid, such as a 11-carboxyundecane phosphonic acid (C-12 phosphonate).
- C-12 phosphonate can be prepared as follows: bromododecanoic acid, 1.12 g (4 mmoles) is dissolved in 10 mL dichloromethane in a 50-mL round-bottom flask. Oxalyl chloride (2 mL of 2M) is added with stirring under a nitrogen atmosphere at room temperature and the reaction is initiated by the addition of 100 ⁇ l N,N'- dimethylformamide (DMF). At one and two minutes after starting the reaction, an additional 100 ⁇ L of DMF is added to the solution. After 15 minutes, 8 mL dichloromethane is added to the reaction mixture and stirring continued under nitrogen for 15 minutes. The solvent is then removed from the acid chloride intermediate with a stream of nitrogen.
- DMF N,N'- dimethylformamide
- the acid chloride intermediate is immediately dissolved in 10 mL dichloromethane and while rapidly stirring, ethanol (350 ⁇ L) and triethylamine (835 ⁇ L) are added.
- the pH of the solution when tested with pH paper is between 7 and 8.
- the solution is stirred for one hour at room temperature.
- the solvent is evaporated off, and the product dissolved in 10 mL hexane and washed with 10 mL water.
- the hexane phase is recovered and evaporated to dryness to recover the ethyl ester intermediate.
- Triethyl phosphite (1.5 mL) is added to the ethyl ester intermediate in a 100 mL round bottom flask and the solution is refluxed under nitrogen.
- the concentration of carboxy- alkyl phosphonates ranges from 0.1 mM-20 mM with 2-5 mM carboxy-alkyl phosphonate preferred to provide sufficient monolayer formation. Suitable self- assembly times can vary from three seconds to 20 hours, with 30 minutes currently preferred.
- the unattached carboxy-alkyl phosphonate is rinsed off the ITO electrodes with three washes of water and the electrodes allowed to dry. If there is insufficient phosphonate, the monolayer may be poorly ordered and the carboxylate group may not be accessible for activation and oligonucleotide probe attachment. An excess of monolayer acts as a barrier to electron transfer by inhibiting the movement of the transition metal complex from the oligonucleotide to the electrode surface. Also, an excess of carboxy-alkyl phosphonate monolayer can lead to electrostatic inhibition of oligonucleotide probe binding and target hybridization.
- the placement of reagents on to the monolayer/ITO electrode of the invention is standardized as known in the art, for example, by marking the electrode on the non- conductive side.
- Example 4 Activation of Monolayer on Electrode.
- the ITO electrode having thereon the monolayer according to Example 3 is exposed to the activating/coupling compounds l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N- hydroxysuccinimide (NHS) in a molar ratio of 4: 1.
- EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
- NHS N- hydroxysuccinimide
- Example 5 Attachment of DNA probe.
- An oligonucleotide probe with an alkyl amine linker on the 3'- or 5 '-end is coupled to the activated monolayer.
- the length of the alkyl amine should be at least three carbons, and preferably between three and twelve carbons with the currently preferred length being six carbons.
- the oligonucleotide probe (20 ⁇ L) at concentrations of 20 to 100 ⁇ M in 1M NaCl/0.25 M NaHCO 3 , pH 9 is pipetted onto the activated monolayer and incubated at room temperature (approximately 25°C) for 30 minutes.
- the oligonucleotide probe solution is removed and the electrode washed by immersion in water followed by washes in 0.1 M sodium phosphate buffer, pH 7, 1.0 M NaCl and water.
- the degree of oligonucleotide probe attachment to the monolayer can be assessed radiochemically by the addition of 32 P-labelled oligonucleotide probe to the reaction mixture.
- Activated carboxyl groups that did not react with oligonucleotide probe are blocked with ethanolamine to reduce non-specific target binding.
- the electrodes are immersed in 0.1 M ethanolamine, pH 8 at 25°C for about 20 minutes. Ethanolamine is rinsed off the electrodes with three washes of water and the electrodes are allowed to air dry.
- oligonucleotide probe concentration of oligonucleotide probe, the pH, incubation time, temperature and blocking agent can vary as would be clear to one of ordinary skill in the art.
- Example 6 Exposure of Electrode to Target Nucleic Acid.
- a nucleic acid target with a nucleic acid sequence that is complementary to a portion of the oligonucleotide probe sequence is allowed to hybridize to the probe.
- a complementary synthetic oligonucleotide that contains 23 guanines is being used.
- the target nucleic acid (20 ⁇ l in 0.8 M NaCl and 0.05 M NaH 2 PO 4 , pH 7.0) is pipetted onto the oligonucleotide probe/monolayer and incubated at 25°C for an hour.
- the target nucleic acid solution is removed and the electrode washed in water followed by washes in 0.1 M NaH 2 PO 4 , pH 7.0, 1.0 M NaCl and water.
- the hybridization conditions may be varied as would be clear to one of ordinary skill in the art.
- the degree of target nucleic acid hybridization to the oligonucleotide probe can be assessed radiochemically by the addition of 32 P-labelled target nucleic acid to the reaction mixture.
- Example 7 Electrochemistry of Electrodes.
- the currently preferred method of electrochemically interrogating the electrodes is cyclic voltammetry, although electrochemical interrogation methods such as chronoamperometry, chronocoulometry and step voltammetry are also useful. Cyclic voltammetry is performed on each ITO electrode as follows. Suitable scan speeds include scan speeds of about 50 mV/s-5000 V/s, with a preferred scan rate of 20 V/s. At this scan rate, there is a maximum signal from bound DNA and a minimum signal from the background. The potential is first swept in the positive direction, with a starting potential of 0 V and switching potential between 1.3 and 1.8 V depending on the scan speed.
- a three-electrode setup is used: a Ag/AgCl reference electrode, a Pt wire auxiliary electrode, and a modified ITO working electrode according to the invention.
- the modified ITO electrode is placed in an electrochemical cell, and 200 ⁇ L of 100 ⁇ M Ru(bpy) 3 2+ in 50 mM sodium phosphate buffer (pH 7.0) is placed above the modified electrode.
- the buffer may contain NaCl (generally up to about 1 M or as desired for the particular system under study) which in some cases increases signal separation.
- the reference electrode and Pt electrode are placed in the electrochemical cell in contact with the Ru(bpy) 3 2+ solution. The sample is interrogated, and the data collected, stored and analyzed.
- FIGs la and lb show the effect of self-assembly time on monolayer formation as indicated by the amount of oligonucleotide probe that the monolayer is able to bind, in picomoles.
- Figure la is for self-assembly times up to 2 hours and
- Figure lb is for times up to 90 hours.
- the amount of oligonucleotide probe bound to the monolayer is an indirect measure of monolayer formation. For self-assembly times up to approximately 10 hours, the self- assembly time does not appear to impact substantially on the ability of the monolayer to bind oligonucleotide probe.
- Monolayer formation was evaluated as a function of the concentration of phosphonate solution to which the electrode is exposed. This evaluation was conducted by examining current above background (current separation) per pmole of guanine of the oligonucleotide strand as shown in Figure 2.
- the monolayers were formed using a 2-hour self-assembly time at all concentrations and electrochemical measurements were made using cyclic voltammetry at 20 V/s and a Ru(bpy) 2+ concentration of 100 ⁇ M.
- the electrochemical response was measured as ⁇ A of peak separation over background per picomole of guanine coupled to the monolayer. Concentrations of the 12-carbon carboxy-alkyl phosphonate in the self-assembly solution ranged from 0.1 to 20 mM. The effect of the concentration of the phosphonate solution was minimal.
- Example 9 Cyclic voltammogram of monolayer.
- Figure 4 shows the dose response of self-assembled monolayer electrodes with different amounts of guanine- containing oligonucleotide attached.
- the amount of oligonucleotide coupled to the monolayer ranged from 0.008 to 0.466 picomoles of oligonucleotide strand on each electrode.
- the oligonucleotide was a synthetic 34-mer with 23 guanines per strand.
- Electrochemical measurements were made using cyclic voltammetry at 20 V/s and a Ru(bpy) 3 2+ concentration of 100 ⁇ M. This figure shows that one can discriminate between monolayers that have differing amounts of oligonucleotide attached.
- the signal (current) is proportional to the number of guanines on each electrode.
- Figure 5 is a graphical representation of Figure 4, where the ⁇ A of signal over background vs. pmole of guanine in the oligonucleotide strand on each electrode is plotted.
- Monolayers were prepared which had varying amounts of oligonucleotide and the electrochemical response ( ⁇ A of peak separation over background) was measured and plotted as a function of the amount of guanine (in picomoles) in the oligonucleotide coupled to the monolayer.
- the graph shows the direct correlation between the electrochemical response and the amount of guanine.
- Example 10 Alternative Method for Immobilizing Oligonucleotides via Carboxy-alkyl Phosphonates.
- An alternative method of forming self-assembled monolayers with attached oligonucleotides is to couple carboxy-alkyl phosphonate to the oligonucleotide prior to formation of the self-assembled monolayer.
- oligonucleotide with an alkyl amine linker on the 3'- or 5 '-end is added to a solution of dimethyl sulfoxide containing 0.005-1 mM carboxy-alkyl phosphonate, 0.2 M 1- ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.05 M N- hydroxysuccinimide (NHS).
- EDC 1- ethyl-3-(3-dimethylaminopropyl) carbodiimide
- NHS N- hydroxysuccinimide
- the reaction mixture may be incubated for 6-8 hours at 25°C Radiolabeled oligonucleotide can be used if the degree of oligonucleotide-phosphonate attachment to the electrode is to be quantitated.
- the reaction mixture (20 ⁇ l) containing the carboxy-alkyl phosphonate oligonucleotide conjugate is pipetted onto an ITO glass electrode and incubated for 2- 4 hours at 25°C to allow the monolayer to form.
- the unattached material is removed and the electrode washed sequentially with water, 0.1 M NaH 2 PO 4 , pH 7.0, 1.0 M NaCl and water.
- this method of coupling the carboxy-alkyl phosphonate to the oligonucleotide yields heterogeneous products that have one or more carboxy-alkyl phosphonate groups attached to the oligonucleotide, the primary attachment site being the alkyl amine on the 3'- or 5 '-end of the oligonucleotide, with other exocyclic amines on the bases serving as secondary attachment sites for the carboxy-alkyl phosphonate.
- Attachment to the ITO electrode and hybridization of target nucleic acid molecules can be impacted by how many carboxy-alkyl phosphonate groups are present on the oligonucleotides.
- oligonucleotide-phosphonate products using oligonucleotides with blocked exocyclic amino groups so that only one carboxy- alkyl phosphonate is attached to each oligonucleotide via the alkyl amine on the 5'- or 3 '-end.
- a carbodiimide in an aqueous-non-aqueous solvent mixture may be used to couple the carboxy-alkyl phosphonate to the oligonucleotide that is immobilized on glass beads after synthesis and still retains protective groups on the exocyclic amines.
- the pure carboxy-alkyl phosphonate oligonucleotide conjugate is preferably attached to ITO electrodes by first dissolving the conjugate in 85-100% dimethyl sulfoxide. Free carboxy-alkyl phosphonate is added to the solution to give a solution of alkyl phosphonate having a concentration of about 5 ⁇ M-5 mM. This mixture (20 ⁇ l) is pipetted onto an ITO glass electrode and incubated for 2-4 hours at 25°C to allow the monolayer to form. The unattached material is removed, and the electrode washed sequentially with water, 0.1 M sodium phosphate, pH 7.0, 1.0 M NaCl, and water.
Abstract
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AT00913366T ATE312200T1 (en) | 1999-04-22 | 2000-02-04 | SINGLE LAYER AND ELECTRODE FOR DETECTING LABLED MOLECULES AND METHOD FOR USE THEREOF |
AU34830/00A AU781085B2 (en) | 1999-04-22 | 2000-02-04 | Monolayer and electrode for detecting a label-bearing target and method of use thereof |
CA002367594A CA2367594A1 (en) | 1999-04-22 | 2000-02-04 | Monolayer and electrode for detecting a label-bearing target and method of use thereof |
DE60024618T DE60024618D1 (en) | 1999-04-22 | 2000-02-04 | Single Layer and Electrode for the Detection of Marked Molecules and Method for their Use |
KR1020017013429A KR20020008157A (en) | 1999-04-22 | 2000-02-04 | Monolayer and electrode for detecting a label-bearing target and method of use thereof |
EP00913366A EP1185692B1 (en) | 1999-04-22 | 2000-02-04 | Monolayer and electrode for detecting a label-bearing target and method of use thereof |
NZ514682A NZ514682A (en) | 1999-04-22 | 2000-02-04 | Monolayer and electrode for detecting a label-bearing target and method of use thereof |
JP2000614433A JP2002542794A (en) | 1999-04-22 | 2000-02-04 | Monolayers and electrodes for detecting label-bearing targets and methods of use |
NO20014882A NO20014882L (en) | 1999-04-22 | 2001-10-08 | Monolayer and electrode for detecting a marker-borne target material and its use |
HK02106722.0A HK1045178B (en) | 1999-04-22 | 2002-09-13 | Monolayer and electrode for detecting a label-bearing target and method of use thereof |
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US10094800B2 (en) | 2002-03-07 | 2018-10-09 | Atlas Genetics Limited | Assays and apparatus for detecting electrochemical active markers in an electric field |
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Also Published As
Publication number | Publication date |
---|---|
AU781085B2 (en) | 2005-05-05 |
NO20014882L (en) | 2001-12-27 |
CN1347460A (en) | 2002-05-01 |
HK1045178A1 (en) | 2002-11-15 |
HK1045178B (en) | 2006-08-11 |
NZ514682A (en) | 2003-07-25 |
EP1185692B1 (en) | 2005-12-07 |
JP2002542794A (en) | 2002-12-17 |
NO20014882D0 (en) | 2001-10-08 |
EP1185692A1 (en) | 2002-03-13 |
US6127127A (en) | 2000-10-03 |
KR20020008157A (en) | 2002-01-29 |
CA2367594A1 (en) | 2000-11-02 |
AU3483000A (en) | 2000-11-10 |
ATE312200T1 (en) | 2005-12-15 |
DE60024618D1 (en) | 2006-01-12 |
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