MXPA01009221A - Electropolymerizable film, and method of making and use thereof - Google Patents

Abstract

An electrode and method of preparing an electrode by electropolymerizing a film on the conductive working surface of an electrode. The electrode is modified by reductive electropolymerization of a thin film of poly[Ru(vbpy)32+]or poly[Ru(vbpy)32+/vba](vbpy=4-vinyl-4'methyl-2,2'-bipyridine and vba=p-vinylbenzoic acid) and the electrode is used for the electrochemical detection of aqueous GMP, poly[G], and the surface immobilized single-stranded DNA probes. The film is formed from a co-polymer of a mediator such as Ru(vbpy)32+ and a functionalized moiety having a carboxylate group such as p-vinylbenzoic acid. A DNA probe is attached covalently to the carboxylate group via a carbodiimide reaction followed by amidation of an amino-linked single-stranded DNA. In the presence of these guanine containing moieties, a dramatic enhancement in the oxidative current for the Ru3+/2+ couple (present in the polymeric thin film) due to the catalytic oxidation of guanine is observed.

Description

ELECTROPOLIMER1ZABLE FILM, AND METHOD FOR DEVELOPING AND USING THE SAME BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to electrodes for detecting nucleic acid hybridization, and methods for making and using said electrodes.

DESCRIPTION OF THE RELATED TECHNIQUE Recent advances in surface modification techniques have facilitated many new methods for bioassay technology, particularly when coupled with sophisticated fluorescent detection technologies. For example, the analysis of gene expression (Schena, M. et al., Science 1995, 270, 467); sequencing of genomic DNA in high density arrays (Chee, M. et al., Science, 1996, 274, 610); and the detection of nucleic acids to identify infectious organisms (Spargo, CA et al., Molecular and Cellular Probes 1993, 7, 395; Martin, WJ in The Polymerase Chain Reaction; Mullis, KB; Ferre, F .; Gibbs, RA, eds., 1994, 406-417, Berkhauser, Boston) have the potential for higher selectivity and sensitivity when compared to pre-existing cultures or immunoassay-based methods. Although these systems present significant advances, they still involve extensive pre-treatment steps and the use of costly fluorescent microscopes. Electrochemical detection of nucleic acids provides an alternative for fluorescent bioassay techniques that potentially eliminate the need to label (Johnston, DH et al., Metal lons Biol. Syst, 1996, 33, 297; Johnston, DH; Cheng CC et al. , Inorg, Chem. 1994, 33, 6388; Steenken, S. et al., J. Am. Chem. Soc. 1997, 119, 617: Johnston, DH et al., J. Am. Chem. Soc. 1995, 117, 8933; and Johnston, DH et al., J. Phys. Chem. 1996, 100, 13837). The invention herein utilizes the discovery in which the guanine nucleobases of polymeric DNA produce an arrangement of active redox labels suitable for ultrasensitive detection which, in conjunction with ultramicroelectrode methods, provide a method for detecting many physiologically relevant nucleic acids prior to PCR amplification. The incorporation of individual microelectrodes in one arrangement allows the production of low-cost, fast-acting devices with multi-density, high-density sensor arrays. Nucleic acids can be detected in solution by means of catalytic oxidation of guanine bases using Ru (bpy) 32+ as the mediator (Johnston, DH et al., Metal lons Biol. Syst, 1996, 33, 297; Johnston, DH et al. al., Inorg, Chem. 1994, 33, 6388; Johnston, DH et al., J.

Am. Chem. Soc. 1995, 117, 8933; and Johnston, D.H. et al., J. Phys. Chem. 1996, 100, 13837). In solution, Ru (bpy) 32+ exhibits a reversible redox torque at 1.05 V similar to the oxidation potential observed for guanine. The addition of a guanine-containing DNA to a solution of Ru (bpy) 32+ leads to a catalytic enhancement in the oxidation stream in accordance with a two-step mechanism: Ru (bpy) 32+? Ru (bpy) 33+ e-Ru (bpy) 33+ + DNA? ADN0X + Ru (bpy) 32+ where ADN0X represents a DNA molecule in which guanine has undergone an oxidation of electrons. As established in the patent of Thorp et al. (US Patent No. 5,871, 918), the hybridized DNA can be immobilized on a solid support, and the oxidation agent then reacted with the hybridized DNA by immobilizing the oxidation agent in the same solid support and immersing the solid support in a solution under conditions sufficient to allow the oxidation-reduction reaction of the oxidation agent and a previously selected base to occur. As mentioned above in the patent of Thorp et al. (U.S. Patent No. 5,968,745) additional schemes in which the DNA was immobilized on poly (ethylenterephthalate) or PET membranes and DNA probes attached directly to the indium-tin oxide (ITO) electrodes were used for the detection of complementary DNA with application as an amplicon bioassay by PCR (Napier, ME et al., Langmuir 1977, 13, 6342; Napier, M. E. et al., H. H. Bioconjugate Chem, 1997, 8, 906). In essence, the body of previous research has focused on two scenarios: 1) mediators of solution with DNA in solution and 2) mediators in solution with immobilized DNA. Electrochemical copolymerization has been used to prepare matrices. For example, a DNA matrix has been prepared on a surface of the electrode by means of electrochemically directed copolymerization of pyrrole and oligonucleotides carrying a pyrrole group, along with radiolabeling of the oligonucleotides to detect hybridization (Livache, T. et al., Nucleic Acids Res. 1994, 22, 2915; Roget, A. et al., Nucleosides &Nucleotides 1995, 14, 943). Also, biosensors have been designed using an electroactive polypyrrole functionalized with an oligonucleotide probe (Korri-Youssoufi, H. et al., J. Am. Chem. Soc. 997, 119, 7388). Although Lívache and Roget use a radioactive label to detect DNA bound to the electropolymerized film and Korri-Yousoufi monitors the potential of the film itself to indirectly detect DNA hybridization, the invention herein allows direct detection of bound DNA to the electropolymerized film by means of a faradaic current from guanine. Thus, the Korri-Yousoufi approach is a potentiometric method, where the method of the present is amperometric. The polymers described herein provide a catalyst for the transfer of electrons from the nucleic acid to the electrode allowing the detection of faradaic current, which must be too slow to provide a practical signal without the immobilized mediator. In addition, the films of the invention are electrochemically inert in the 0-0.9 V region, wherein the above polypyrrole films are reactive in this region. The Yachynych patent (U.S. Patent No. 5,540,828) provides a method for making electrodes for protein recognition by oxidative electropolymerization. In contrast to Yachynych, the invention herein is used for nucleic acids, in the invention the polymers are reductive rather than oxidatively formed during polymerization, the invention uses vinyl-containing polymers, and the metal complex used is both initiator for electropolymerization and immobilized mediator. It is therefore an object of the present invention to provide electrodes modified on its surface for the detection of electron transfer events in potentials close to those observed for guanine or other preselected bases in DNA or RNA. It is another object of the invention to immobilize a mediator, mainly the Ru2 + center, to be used in the electrochemical detection of guanine bases present both in solution and in species immobilized on its surface. It is another object of the invention to use electropolymerization to generate electrodes modified with probes that give a greater oxidation current at the time of hybridization with targets containing large numbers of pre-selected bases. It is another object of the invention to provide an agent that acts both as an initiator for electropolymerization and as a meter for oxidation of a preselected base. Other objects and advantages will be apparent from the following description and appended claims.

BRIEF DESCRIPTION OF THE INVENTION The invention herein utilizes a modified metal complex so that polymerization occurs under defined circumstances, and includes an electrode and a method for preparing an electrode by electropolymerizing a film on the conductive work surface of an electrode. The electrode is modified by reductive electropolymerization of a thin film of poly [Ru (vbpy) 32+] or poly [Ru (vbpy) 327 vba] (vbpy = 4-vinyl-4'methyl-2,2'-bipyridine and vba = p-vinylbenzoic acid) and the electrode is used for the electrochemical detection of aqueous GMP, poly [G], single-stranded DNA probes immobilized on its surface, and hybridized DNA or RNA targets. The film is formed from a co-polymer of a mediator such as Ru (vbpy) 32"and a functionalized portion having a carboxylate group such as p-venylbenzoic acid.A DNA probe is covalently bound to the carboxylate group by means of a carbodumide reaction followed by the amidation of a single chain DNA linked to an amino In the presence of these guanine-containing portions, a dramatic improvement in the oxidative current is observed for the pair Ru3 + 2+ (present in the film polymeric thin) due to the catalytic oxidation of guanine Other objects and characteristics of the invention will be apparent from the following description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A and 1B are cyclic voltammograms showing the electropolymerization of (A) poly [Ru (vbpy) 32+], and (B) 5: 1 of pol [Ru (vbpy) 32 + / vba] from an acetonitrile solution containing 0.1 M TBAH in a glass-like carbon electrode (scanning speed 10 mV / s, Ag / AgNO3 reference.) The concentration of Ru (vbpy) 32"in solution was 0.2 mM. Figure 2 shows cyclic voltammograms showing the oxidation of guanosine monophosphate (GMP) using (A) an unmodified GCE and a modified CGE with poly film [Ru (vbpy) 32+] in the (B) absence and (C) presence of GMP (50 mV / s scanning speed, Ag / AgCl reference, pH regulated solution with 50 mM phosphate, pH 7.0) Figure 3A shows the first and second oxidative scans of a film modified GCE of poly [Ru (vbpy) 32+] and Figure 3B shows the first and the second oxidative scans of a modified GCE with poly film [Ru (vbpy) 32+] in the presence of GMP ( 50 mV / s sweep rate, Ag / AgCl reference, pH regulated solution with 50 mM phosphate, pH 7.0). Figure 4 shows cyclic voltammograms showing the oxidation of poly [G] using (A) an unmodified GCE and a poly modified [Ru (vbpy) 32+] in (B) the absence and (C) the presence of po! i [G] (scanning speed of 50 mV / s, reference Ag / AgCI, solution regulated in its pH with 50 mM phosphate, pH 7.0). Figure 5 is a cyclic voltammogram of 5: 1 of a GCE modified with polyJRuívbpyJs2 * film} with CpFe (CsH4-NH2) immobilized (scanning speed 50 mV / s, reference Ag / AgN03, 0.2 mM Ru2 +, 0.1 M TBAH solution). Figure 6 shows cyclic voltammograms of (A) 5: 1 GCE modified with poly [Ru (vbpy) 32 + / vba] film with 20 poly [dG] elements immobilized at pH 6.5, and (B) 5: 1 of modified GCE with poly [Ru (vbpy) 32+ vba] film with 20 elements of immobilized poly [dG] at a pH of 9.0 (scanning speed of 50 mV / s, reference Ag / AgCl, regulated solution in its pH with phosphate at 50 mM, pH 7.0). Figure 7 is a representation of scheme 2 showing the immobilization of the DNA probe bound to amino on its surface.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED MODALITIES OF THE INVENTION The invention of the present invention relates to a method for making an electropolymerizable film and the resulting film and the use thereof. In general, the method of preparing the electrode of the invention having therein the electropolymerizable film of the invention, comprises: (a) electropolymerizing a film in an electrode, said film comprising a co-polymer of a metal complex that acts both as an electropolymerization initiator as an electrotransfer mediator and a functionalized portion having a carboxylate group; and (b) attaching a DNA probe covalently to the carboxylate group by means of a reaction with carbodiimide followed by amidation of a single chain DNA linked to an amino. The electrode of the invention is useful for the electrochemical detection of aqueous GMP, poly [G], and nucleic acids immobilized on its surface containing a preselected base. It comprises a substrate having a conductive working surface modified by reductive electropolymerization of a thin film. The thin film is selected from the group consisting of poly [Ru (vbpy) 32+] and poly [Ru (vbpy) 32 + / vba], where vbpy is 4-vinyl-4'methyl-2,2 ' -bipiridine and vba is p-vinylbenzoic acid.

In particular, the present invention provides thin polymeric films containing polypyridyl Ru "complexes, generally based on Ru (vbpy) 32+, preferably prepared by reductive electropolymerization in Pt and vitreous carbon electrode surfaces from dilute acetonitrile solutions (vbpy = 4-vinyl-4'-methylbipyridine) These films present a pair of oxidative redox at 1.1 V (all potentials vs. Ag / AgCl), slightly above that observed for guanine (1.05 V vs. Ag / AgCl) in aqueous solution (Abruna, HD et al., J. Am. Chem. Soc. 1981, 103, 1; Denisevich, P. et al., Inorg. Chem. 1982, 21, 2153). , therefore, are active catalysts for the electrooxidation of guanine and polymers containing guanine or other preselected bases, such as DNA or RNA, and the electropolymerization of a mixture of Ru (vbpy) 32+ and p-vinylbenzoic acid (vba). is used to produce movies that which contain the ruthenium catalyst and to which the oligonucleotides attached to an amine can be attached by means of a reaction with carbodiimide which marks the vba. As part of this invention, Ru-modified (vbpy) 32 films are used to catalyze the oxidation of DNA and Ru (vbpy) 32"and vba copolymers are used to prepare loci specifically assembled in place for detection of DNA Amplification To the extent that methods using an electrode having an electropolymerized membrane in accordance with the present invention involve contacting the nucleic acid sample with an oligonucleotide probe to produce a hybridized DNA or RNA, it may be desired for certain applications Amplify the DNA or RNA before putting them in contact with the probe. The amplification of a nucleic acid sequence, selected or targeted can be carried out by any suitable means, such as those described and discussed in the copending applications.

Nucleic acid detection As noted above, the invention herein includes an electrode in which the electropolymerized membrane has been formed, and methods for using this electrode allow the detection of hybridized nucleic acid. In this method, a nucleic acid sample is contacted with an oligonucleotide probe to form hybridized nucleic acid. Oligonucleotide probes that are useful in the methods of the present invention can be any probe comprising between about 4 or 6 bases to about 8 or 100 bases or more, more preferably between about 8 and about 30 bases. Oligonucleotide probes can be prepared having a wide variety of base sequences in accordance with techniques that are well known in the art. Suitable bases for preparing the oligonucleotide probe can be selected from nucleotide bases as they occur in nature such as adenine, cytosine, guanine, uracil, and thymine; and as they do not occur in the nature or bases of "synthetic" nucleotides such as 8-oxo-guanine, 6-mercaptoguanine, 4-acetylcytidine, 5- (carboxyhydroxyethyl) uridine, 2'-O-methylcytidine, 5-carboxymethylamino-methyl -2-touridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-0-methylpseudouridine, D-galactosylkeosine, 2'-O-methylguanosine, inosine, 7-deazaguanine, N6 -sopentenyldenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethy1- 2-thiouridine, D-mannosylkeosine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N 6 -sopentenyldenosine, N- (9-D-ribofuranosyl-2-methylthiopurine-6-yl) carbamoyl) threonine, N- ((9-D-ribofuranosylpurine-6-yl) N-methyl-carbamoyl) threonine, uridine-5-oxyacetic acid methyl ester, uridine-5-oxyacetic acid, wibutoxin, pseudouridine, kerosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 5-methyluridine, N - ((9-D-ribofuranosylpurine-6-yl) carbamoyl ) threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, wentsin, and 3- (3-amino-3-carboxypropyl) uridine. Any oligonucleotide base structure can be employed, including sugars modified with DNA, RNA (although DNA is more preferred than RNA), such as carbocycles, and sugars containing 2 'substitutions such as fluoro and methoxy. Oligonucleotides can be oligonucleotides in which at least one or all of the phosphate residues forming intemucleotide bridges are modified phosphates, such as methylphosphonates, methylphosphonothioates, phosphoromorpholidats, phosphoropiperazidates and phosphoroamidates (for example each phosphate residue forming different internucleotide bridges can be modified as described). The oligonucleotide can be a "peptide nucleic acid" as described in P. Nielsen et al., 1991, Science 254, 1497-1500. The only requirement is that the oligonucleotide probe possesses a sequence, wherein at least a portion of which is complementary to a known portion of the target nucleic acid sequence. It is desired 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 to two or more probes in a "sandwich" assay).

Preselected base After hybridization, the hybridized nucleic acid that binds to the electropolymerized membrane can be reacted with a suitable mediator that is immobilized to the membrane and is capable of oxidizing a preselected base in an oxidation-reduction reaction. The preselected base can be any such as it occurs in nature or a synthetic nucleotide base that undergoes oxidation at the time of reaction with the selected mediator. The preselected base should exhibit unique oxidation rates when pairing with each of the four bases as they occur in nature. Generally, bases whose 5'-mononucleotides (eg 5'-dexosyribonucleotide or 5'-ribonucleotide) have constant rates above 104M "V1 can be detected using the catalytic reaction Examples of suitable preselected bases include but are not limited to guanine, adenine, 8-oxo-guanine and 8-oxo-adenine, 8-bromo-guanine, janthine, 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-dimethylalil-purine, 2-thioadenine, 8-hydroxyadenine, 8-methoxyadenine Typically, the preselected base is selected from the group consisting of guanine, adenine, 6-mercaptoguanine, 8-oxo- guanine, and 8-oxo-adenine, guanine being the preselected base as it is presented in the actuality nature Preferred and 8-oxoguanine being the currently preferred synthetic pre-selected base.

Mediator The mediator that can be used to allow electron transfer and to initiate electropolymerization is one that (a) has a reversible oxidative redox pair and is capable of oxidizing a preselected base, (b) has a substituent capable of undergoing electropolymerization in potentials other than those of the oxidative redox pair, and (c) presents a different redox pair that can be used to initiate electropolymerization. For example, the ruthenium2 + (2,2'-bipyridine) 3 (Ru (vbpy) 32+ complex where vbpy = 4-vinyl-4'methyl-2,2'-bipyridine) exhibits an oxidative redox pair at about 1.0 V, which can mediate guanine oxidation, the vinyl group in the bpy ligand can be used for polymerization, and the complex shows a reductive redox couple at -1.1 V which can be used to initiate the polymerization. Mediators that meet these criteria include metal complexes containing vinyl-substituted poly-pyridyl ligands that exhibit both oxidative and reductive redox pairs.

Oxidation-reduction reaction detection The occurrence of the oxidation-reduction reaction can be detected using an electropolymerized membrane in an electrode according to the present invention to observe a change in the electronic signal that is indicative of the oxidation reaction event -reduction. Typically, an electrode modified with the electropolymerized film containing the mediator and to which the nucleic acid is immobilized is contacted with a solution that is also in contact with a reference and auxiliary electrode (with most of the current going through through the auxiliary electrode). Likewise, suitable reference electrodes will also be known in the art and include, for example, silver / silver chloride electrodes. The detection of the electronic signal related to the oxidation-reduction reaction allows the determination of the presence or absence of hybridized nucleic acid. The step to determine the presence or absence of the hybridized nucleic acid typically includes (i) measuring the reaction rate of the oxidation-reduction reaction, (ii) comparing the measured reaction rate with the oxidation-reduction reaction rate of the complex of the transition metal with a single-stranded nucleic acid, and subsequently (iii) determining whether or not the measured reaction rate is essentially the same as the oxidation-reduction reaction rate of the transition metal complex with a single-stranded nucleic acid. The step of measuring the reaction rate can be carried out by any suitable means. For example, the relative reaction rate can be determined by comparing the current on the same scan scale, probe concentration, target concentration, mediator, pH regulator, temperature, and / or electrochemical method. The oxidation-reduction reaction rate can be measured according to means known to those skilled in the art. Typically, the oxidation-reduction reaction rate is measured by measuring the electronic signal related to the occurrence of the oxidation-reduction reaction. For example, the electronic signal related to the oxidation-reduction reaction can be measured by providing a suitable apparatus in electronic communication with an electrode coated with an electropolymerized membrane as described herein. A suitable apparatus is a potentiostat capable of measuring the electronic signal that is generated to provide a measurement of the oxidation-reduction reaction rate of the reaction between the hybridized nucleic acid and the mediator. The electronic output may be characteristic of any electrochemical method, including cyclic voltammetry, normal pulse voltammetry, chronoamperometry, and square wave voltammetry, cyclic voltammetry being preferred today. A computer as is known in the art can be used to control the use of the electrode and to record results of such use. The method that is most frequently used to electropolymerize membranes in ITO electrodes according to the invention is cyclic voltammetry. In cyclic voltammetry, the potential of the electrochemical system varies linearly from an initial potential (0-800 mV) to a final potential (1300-1800 mV). When the final potential is reached, the sweep direction is inverted and the same potential scale is swept in the opposite direction. The potential varies at a constant sweep speed (25 mV / s at 50 V / s). For most experiments, the initial potential is set at 0 mV and the final potential is sufficient to effect oxidation of the mediator. The currently preferred scanning speed is 50 mV / s with a change potential of 1.4 V. The current is collected at each potential and the data is marked as a current against the potential spectrum. As an alternative to cyclic voltammetry, potential step methods such as chronoculomimetics or chronoamperometry can be used to analyze the electropolymerizable membranes of the invention. In cronoculomolytry, a potential step is applied. Starting at the initial potential (0 mV-800 mV), the electrochemical system is brought directly to the final potential (1100 mV-1600 mV). The electrochemical system is kept in the final potential for a specific time (50 μs to 10 s) and the load is collected as a function of time. Although not done at present, if desired, the potential can be placed back to the initial potential and the load can be collected at the initial potential as a function of time. In chronoamperometry, the electrochemical system is placed from an initial potential (0 mV-800 mV) directly to a final potential (1000-1500 mV) during a specific period (50 μs to 10 s) and the current is collected as a function of time. If desired, the potential can fall back to the initial potential, and the current can be controlled at the initial potential as a function of time. In the methods described in the patent of Thorp et al. (U.S. Patent No. 5,871, 918), metal complexes are used to obtain an electrochemical current from single and double stranded nucleic acids. Preset bases such as guanine provide an electrochemical signal, and this signal is much weaker for double-stranded DNA. Said methods usefully exhibit high structural sensitivity, and can solve a simple base non-pairing. Said methods are therefore particularly useful for DNA sequencing. However, two disadvantages of such methods are: (a) there is a negative signal going from the probe string to the hybrid, and (b) the number of pre-selected bases is limited which limits the signal. The techniques set forth herein provide solutions to these problems. In addition, these techniques are particularly useful for diagnostic assays, and are particularly useful for the quantitative detection of nucleic acids. In view of the foregoing, it is also described herein and in the patent of Thorp et al. (U.S. Patent No. 5,871, 918), a method for detecting the presence or absence of a target nucleic acid in a test sample suspected of containing it, wherein the target nucleic acid contains at least one preselected base. In this method, the preselected base is located in the target nucleic acid, instead of being in the oligonucleotide probe.

Test samples The method can be carried out on a test sample containing the target nucleic acid. Any test sample suspected to contain the target nucleic acid can be used, including, but not limited to, tissue samples, such as biopsy samples and biological fluids such as blood, saliva, urine and semen samples, cultures of bacteria, soil samples, food samples, etc. The target nucleic acid can be of any origin, including animal, plant or microbiological (eg viral, prokaryotic and eukaryotic organisms, including bacteria, protozoa, and fungi, 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, food specimens, dental specimens and veterinary specimens. The sample may be processed or purified before carrying out the present method in accordance with techniques known or apparent to those skilled in the art; and nucleic acids can be digested, fragmented, and / or amplified (see above) before carrying out the present method, if desired.

Detection method Detection of the preselected base in a target nucleic acid using an electrode with an electropolymerized membrane according to the invention in the present invention comprises (a) contacting the test sample with an oligonucleotide probe that specifically binds to the target nucleic acid to form a hybridized nucleic acid, (b) detecting the presence or absence of the oxidation-reduction reaction related to the hybridized nucleic acid; and (c) determining the presence or absence of the target nucleic acid in the test sample from the oxidation-reduction reaction detected in the preselected base. The oligonucleotide probe can be immobilized on a solid support (the electropolymerized film) to facilitate the separation of the test sample from the hybridized nucleic acid, the separation step being first and then the detection step (e.g., between steps (a) and (b) or between steps (b) and (c)). Alternatively, the oligonucleotide probe can be provided free in solution, and other means can be provided for separating the hybridized nucleic acid from the sample (eg, by means of a mediating nucleic acid that binds to the oligonucleotide probes , or by a biotin-avidin binding interaction where the biotin binds to the oligonucleotide probe and the avidin is immobilized on a solid support). The oxidation-reduction reaction, and any step after the detection step can be performed in the electropolymerized film before or after the film is contacted with the conductive working surface of the substrate. Preferably, the target nucleic acid contains at least more than ten preselected bases than the oligonucleotide probe, or more preferably at least more than 50 or 100 of the preselected base than the oligonucleotide probe. A greater current improvement is obtained when the target nucleic acid contains many more preselected bases than the oligonucleotide probes.

Optionally, but preferably, the oligonucleotide probe is free from the preselected base, or is at least essentially free from the preselected base (i.e., contains sufficiently less than the preselected base so that the probe signal does not interfere with or it is not confused with a target nucleic acid signal). Where a sequence of bases is not available as they occur in nature that will conveniently hybridize to the target nucleic acid, the strategy of using alternative bases that are inactive to redox (discussed above) can be employed. The target nucleic acid is preferably greater than the oligonucleotide probe, and at least one of the preselected bases is "pending", ie it does not hybridize to the oligonucleotide probe in the hybridized nucleic acid. Preferably, at least 10, 50 or 100 of the preselected bases are "pending" bases thus providing substantial amplification of the detected electrochemical signal. For example, an oligonucleotide probe can be chosen that does not contain any guanine residue (for example only A, T, and C). The cyclic voltamogram of Ru (bpy) 32+ in the presence of this chain is very similar to that which does not have the oligomer. This probe is then hybridized to a guanine containing target chain either in regions of overlapping base pairs and / or in slopes if the target nucleic acid is larger than the oligonucleotide probe. Since multiple guanines are detected, the signal is amplified in relation to the number of hybrids formed.

In a case where genomic DNA or RNA is a target chain, large numbers of pending guanines are found, which will give a tremendous signal amplification. For example, in a preferred embodiment, the assay for the preselected base in the target chain involves immobilization of the probe chain (preferably redox-inactive) in the film oriented near the electrode surface, which provides a backup signal lower when it is swept. The electropolymerized film is then contacted with a solution of the target chain, which contains the preselected base. If hybridization occurs, the target chain will be in close proximity to the electrode, and an improvement in current will be detected. This method is particularly well suited to the quantitative detection of nucleic acids, since the greater the extent of hybridization, the greater the amount of base preselected on the surface, and the greater the electrochemical signal.

Alternative bases that are inactive of redox An alternative base that can substitute guanine (ie, a base which, like guanine, has a higher binding affinity for cytosine than other bases in a nucleic acid duplex), can be used. in the probe chain but it will not be oxidized by the mediator under the applicable reaction conditions. When the preselected base in the target nucleic acid is guanine and the nucleic acid also contains cytosine (which is ordinarily linked with guanine in the probe), then the probe contains an alternative base that binds to the cytosine in the hybridized nucleic acid. The alternative base can, for example, be inosine. Inosine is three orders less magnitude reactive than guanine. The reaction step typically comprises reacting the transition metal complex with the nucleic acid under conditions sufficient to effect selective oxidation of the preselected base is to oxidize the alternative base. Thus, a method for detecting a target nucleic acid, wherein the target nucleic acid contains at least one preselected base and the capture probe or nucleic acid contains inactive alternative redox bases comprises: (a) contacting the nucleic acid target with a complementary nucleic acid that specifically binds to the target nucleic acid to form a hybridized nucleic acid; (b) detecting the oxidation-reduction reaction, and (c) determining the presence or absence of the nucleic acid from the oxidation-reduction reaction detected in the preselected base. The features of the present invention will be clearly understood by reference to the following examples, which are not intended to be limiting of the invention.

EXAMPLES EXAMPLE 1 Reagents and DNA The inorganic reagents used in these experiments were of analytical grade or higher. The inorganic complex, [Ru (vbpy) 3] (PF6) 2, was prepared using standard literature procedures (Abruna, H.D. et al., J. Am. Chem. Soc. 1981, 103, 1). The detection probe, CpFe (C5H4-C2H4NH2) was synthesized by a reduction of standard LiAIH4 of cyano-substituted ferrocene, CpFe (CX5H4-CH3CN) followed by working-up from diethyl ether. Water-soluble carbodiimide (WSC, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), DCC (dicyclohexylcarbodiimide), TBAH (tetrabutylammonium hexafluorophosphate), NHS (N-hydroxysuccinimide), GMP (disodium guanosine monophosphate salt), poly [G], and vba were purchased from Aldrich (Milwaukke, Wl) and used as received . Two vba recrystallizations were made from 50% ethanol before the electrochemical experiments to ensure purity. MES (sodium salt of 2-morpholinoethanesulfonic acid and 2-morpholinoethanesulfonic acid monohydrate) were purchased from Fluka (New Ulm, Switzerland). Na2HPO4, NaH2PO4, NaCl, and acetonitrile were obtained from Mallinckrodt (Phillipsburg, NJ). The acetonitrile was dried on activated molecular sieves before being used in electropolymerization experiments. Synthetic oligonucleotides were synthesized by the pathology department, The University of North Carolina at Chapel Hill, and purified using 3-micron Amicon concentrators, with a cut-off of 3000 molecular weight. The water was obtained from a Milli-Q Plus purification system (Millipore, Bedford, MA). The vitreous carbon electrodes (GCE, 3 mm diameter) were purchased from BAS (West Lafayette, IN) and polished before use.

EXAMPLE 2 Electrochemical analysis Cyclic voltammograms were collected using a PAR 273A galvanostat / potentiostat. Experiments performed on acetonitrile used a two compartment voltammetric cell equipped with a vitreous carbon working electrode, a platinum mesh counter electrode, and an Ag / AgNO3 reference electrode. The aqueous experiments were performed using a single compartment voltammetric cell equipped with a vitreous carbon working electrode, a platinum wire counter electrode, and an Ag / AgCI reference electrode. Before use, all GCEs with diamond polishing compound METADI (Buehier, Lake Bluff, IL) and AI2O3 (0.5 μm in H2O) were polished thoroughly on a polishing felt platform. The electrodes were subsequently rinsed many times with Milli-Q water and dry acetonitrile immediately before use. The electropolymerization reactions were performed by filling the working electrode compartment with 3.5 ml of [Ru (vbpy) 3] (PF6) 2 at 0.2 mM, a solution of acetonitrile of TBAH at 0.1 M and reductively flushed 10 times between -0.9 and -2.0 V with a sweep speed of 100 mV / s. These solutions must be completely dried and degassed before reductive scanning, and the reference electrode compartment must be filled with 0.1 M TBAH in acetonitrile. For the formation of vba doped films, it was found that a 5: 1 ratio of a solution of [Ru (vbpy) 3] (PF6) 2, to vba, produced films with the highest reproducibility. The electrochemical oxidation of aqueous GMP, poly [G] and DNA probes bound in a pH regulated solution with phosphate at 50 mM, pH 7.0, positively swept from 0.0 to 1.4 V at a scanning speed of 50 mV / s.

EXAMPLE 3 DNA binding to film modified electrodes The binding of the DNA probe was carried out using a standard amidation process in which the surface carboxyl groups (present in the film as vba separators) are activated using well understood carbodiimide chemistry and subsequently undergo amidation reactions with DNA of single chain linked to an amino (Millan, KM et al., Anal, Chem. 1993, 65, 2317; Sehgal, D. et al., Anal. Biochem., 1994, 218, 87). After electropolymerization of poly [Ru (vbpy) 327 vba] in a GCE, the electrode was carefully rinsed with acetonitrile to remove residual [Ru (vbpy) 3] (PFβ), vba, TBAH. The electrode was subsequently inverted and a drop of 50 μl of EDC / NHS solution (made by dissolving 10 mg of EDC and 1 mg of NHS in 1.0 ml of Milli-Q water) was carefully placed on the surface of the electrode. The electrode was covered with an inverted beaker for 30 minutes. The treated GCE was subsequently rinsed numerous times with water and carefully dry. Again, the electrode was inverted, and a drop of 25 μl of a 5 μM DNA probe solution, regulated at its pH, was placed (20 elements of, polifdG) with a 3 '- (CH2) binding group. 6NH3) on the face of the electrode. The electrode was allowed to stand covered and undisturbed for 90 minutes before rinsing with 800 mM NaCl, pH regulated solution with 50 mM phosphate, (pH 7.0). A solution with a high salt content was necessary for this rinsing step to interrupt any electrostatic interaction between the polymer surface and the unbound DNA covalently.

EXAMPLE 4 Quantification of the immobilized probe The 20-element probe was labeled 5 'with -32P using T4 polynucleotide kinase and? -32P-ATP (6000 Ci / mmoles) in accordance with standard procedures (Maniatis, T. et al., In Molecular Cloning: A Laboratory Manual 1989. Cold Spring Harbor Press). The unreacted ATP was removed from the labeled probe using a NucTrap column of Stratagen using standard techniques. The radiolabelled probe was ligated using the identical procedure described for the unlabelled probe using a DNA supply solution (total volume of 270 μl) containing 5 pmoles of labeled probe diluted at 5 μM with unlabelled probe in either MES solution pH 6.5 or solution regulated in its pH with carbonate, pH 9.0. After immobilization, the probe-modified film was mechanically removed from the face of the electrode by rubbing the polymer on a piece of filter paper. The control films were obtained in an identical manner by excluding the ADC / NHS amidation step from the reaction. The radioactivity of these samples was subsequently determined in triplicate using both liquid scintillation and phosphorus formation techniques.

EXAMPLE 5 Preparation of polymer modified electrodes The electropolymerization of poly [Ru (vbpy) 32+] films on electrode surfaces has been well characterized and the films can be manufactured rapidly in reproducible thicknesses using varying scan times and scan speeds as discussed herein. Figures 1A and 1B show polymer growth for both a film containing simple ruthenium, poly [Ru (vbpy) 32+] and a doped film with a vba group containing carboxyl, Scheme I shown shows the formation of the copolymer at the time of electropolymerization. As previously demonstrated, poly [Ru (vbpy) 3 +] films can be reversibly oxidized in media where Ru3"stabilizes at the time of formation (ie, dry acetonitrile and strong acid solutions) (Abruna, HD et al., J. Am. Chem. Soc. 1981, 103, 1, Denisevich, P. et al., Inorg. Chem. 1982, 21, 2153.) Identical behavior was also observed during these studies for contaminated films. of poly [Ru (vbpy) 32 + / vba].

SCHEME I + EXAMPLE 6 Detection of GMP v polilOI The GCE modified with a poly film [Ru (vbpy (32+) is oxidatively partitioned in the presence and absence of GMP and poly [G] in a phosphate pH regulated solution, pH 7.0 (Figures 2, 3 and 4 In Figure 2, the exposure of an unmodified GCE to a 1.0 mM GMP solution produces significantly lower oxidation currents than those of the polymer-modified electrodes, even the subtraction of the background current produced by polymer oxidation. by itself shows positive current indicative of a catalytic electron transfer procedure.An additional support for the proposed catalytic mechanism is shown in Figures 3A and 3B.The voltammogram in Figure 3 demonstrates the stability of the film in the presence of the donor GMP. of electrons In the absence of an adequate concentration of the electron donor, the complete decomposition of the ruthenium centers in the film is observed (Figure 3A). Rgo, when GMP is present in solution, the oxidized Ru3 present in the rigid film is sometimes able to extract an electron from a proximal GMP molecule before rapid decomposition. This effect is observed in the presence of a GMP oxidation wave at the time of the completion of a second oxidative sweep in the GMP solution (Figure 3B). The GMP molecules are too long to diffuse through holes produced at the time of loss of Ru3"by decomposition of the film In an experiment where a poly [Ru (vbpy) 32" film was oxidized in the absence of GMP solution followed by a second oxidative scavenging in a solution containing GMP, a voltammogram identical to that of the second scan was observed in Figure 3A. A separate observation noted that dilution of GMP solutions by two orders of magnitude (0.01 mM) had little effect on the amount of current produced during the first oxidative scan. This effect can be attributed to the electrostatic attraction between the positively charged polymer surface and the anionic GMP. This presumably creates a local concentration on the surface of the electrode that is much greater than that of the bulk solution. The cyclic voltammograms of the oxidation of poly [G] using clean electrodes and modified with poly [Ru (vbpy) 32+] are shown in Figure 4 and demonstrate two definable characteristics. First, no electrochemical oxidation of poly [G] occurs in the absence of a poly [Ru (vbpy) 32+ film. Due to the slow diffusion of said large polymer towards the film (in relation to the cyclic voltammetric time scale), the oxidation of the poly [G] is not detected. However, in the presence of an electrode treated with poly [Ru (vbpy) 32+], an improvement in current is observed over that observed for the oxidation of poly [Ru (vbpy) 32+] alone. Again, the electrostatic attraction of the cationic polymeric surface aids in the same way the diffusion of the large poly [G] molecule to the surface of the electrode where Ru-mediated catalytic oxidation can occur.

EXAMPLE 7 Detection of immobilized probes The ferrocene probe modified with amino (CpFeíCsHU-C2H4NH2)) was attached to a pofi [Ru (vbpy) 32 + / vba] modified electrode of 10-scans (Figure 1B), using the same protocol for aqueous amidation with DCC in methylene chloride substituted with EDC. This activation step was subsequently followed by the amidation of the ferrocene amino group (Figure 5) producing ferrocene molecule immobilized on its surface. The measurable current was observed at 0.2 V in acetonitrile solution and 0.1 M TBAH, representative of the presence of ferrocene pair Fe ^ / Fe3 * on the surface of the modified electrode. Immobilization of the DNA probe bound to an amino was performed on the surface (scheme 2 shown in Figure 7). The immobilization reactions were carried out at two different pH values (6.5 and 9.0) to chemisorb the probe in two different modalities. At high pH, amidation preferably occurs in the primary amine of the group of (CH2) ßH2 attached opposite to any native endogenous amine groups on purine and pyrimidine rings of DNA. The amidation of these native amine groups will produce electrode surfaces where the DNA can bind not only to the amino-linking group, but in many of the native amines as well as produce a surface with few probe molecules "fixed" to the surface (scheme 3). With this assumption, it was thought that the amidation at a pH of 9.0 would produce films containing higher numbers of immobilized DNA probe chains while the stacks would be maximized and the amidation of the native (or linked) amine would be minimized . The films treated with DNA probes at both pH values were subsequently oxidized and compared with currents produced by the film oxidation alone. In Figure 6, it is clear that only those films treated with the DNA probe at a pH of 6.5 produced currents with detectable catalytic improvements. It was only in these films in which the DNA probe was reacted at a pH of 6.5 that showed improvement of catalytic current (typically 8-3 μA of improvement was observed).

EXAMPLE 8 Quantification of the immobilized probe Based on the electrochemical data, catalytic enhancements in oxidation current produced by poly [Ru (vbpy) 32 + / vba] films in the presence of the 20-element G-probe, indicated that approximately a load of more than 10 μC or more of 10"10 moles of electrons (determined by the integration values of voltammograms) are transferred when compared to the oxidation of the polymer alone.The radiolabelled probe was also used as a method to quantify the amount of DNA probe chymosorbed to a surface of GCE at a pH of 6.5 and 9.0 Scintillation counts for surfaces treated with a probe at a pH of 6.5 and 9.0 suggested that 7.4 x 10"12 and 3.0 x 10" 14 moles had been immobilized respectively. Separate labeling experiments were performed using a screen for phosphorus formation images for detection.The values obtained for the immobilized DNA probe were 8.0 x 10"12 and 8.0 x 10"13 moles at a pH of 6.5 and 9.0 using this more alternative and sensitive technique. Based on the electrochemical measurements, it will be necessary to oxidize 10"10 moles of guanine to observe a catalytic improvement of 10 μC Compared to the radiolabelled data, this should correspond to the oxidation of about 50% of the guanine bases immobilized on the surface .

EXAMPLE 9 Detection of hybridized nucleic acid An electropolymerized film is prepared as in the previous examples by means of the polymerization of Ru (bpy) 3 + and vba. An oligonucleotide probe containing an alternative base in place of guanine is bound to the film by means of a carbodiimide reaction. The electrode is then placed in contact with a solution containing a target nucleic acid containing guanine and is complementary to the immobilized probe. After hybridization, the electrode is analyzed by cyclic voltammetry when sweeping at 50 mV / s from 0 to 1.4 V. The electrochemical current is considered higher than for an electrode that has not hybridized to the complementary guanine-containing sequence.

Claims (8)

NOVELTY OF THE INVENTION CLAIMS

1. - An electrode useful for the electrochemical detection of a nucleic acid, comprising a thin film containing a metal complex capable of acting both as an electropolymerization initiator and as a mediator for the oxidation of a preselected base in the nucleic acid and a portion functionalized to which the nucleic acid can be bound.

2. The electrode according to claim 1, further characterized in that the metal complex is selected from the group consisting of polilRuívbpy ^ 24] and poly [Ru (vbpy) 32 + / vba], where vbpy is 4 -vinyl-4'-methyl-2,2'-bipyridine and vba is p-vinylbenzoic acid.

3. A method for preparing an electrode useful for the electrochemical detection of nucleic acids comprises: a) electropolymerizing a film on an electrode, said film comprising a co-polymer of a mediator and a functionalized portion having a carboxylate group; and b) attaching an oligonucleotide probe covalently to the film.

4. The method according to claim 3, further characterized in that the oligonucleotide probe is joined by means of a carbodiimide reaction followed by the amidation of a single chain DNA linked to an amino.

5. The method according to claim 3, further characterized in that the mediator is Ru (vbpy) 32+ and the functionalized portion is p-vinylbenzoic acid.

6. An electrode useful for the electrochemical detection of a preselected base in a nucleic acid, said electrode comprises: a) a substrate having a conductive work surface; and b) an electropolymerized film on said conductive work surface, said electropolymerized film comprises a co-polymer of a mediator and a functionalized portion having a carboxylate group.

7. The electrode according to claim 6, further characterized in that the mediator is Ru (vbpy) 32+ and the functionalized portion is p-vinylbenzoic acid.

8. A method for determining the presence of a target nucleic acid in a sample, comprising: a) contacting an electropolymerized film comprising a co-polymer of a mediator and a functionalized portion having a carboxylate group, with a sample which is suspected to contain the target nucleic acid and an oligonucleotide probe, such that the nucleic acid and the oligonucleotide probe form a hybridized nucleic acid in the film; b) detecting the oxidation-reduction reaction; and c) determining the presence or absence of the nucleic acid from the oxidation-reduction reaction detected.