WO2008048216A2 - Electrocatalytic detection of carbon nanotubes - Google Patents

Abstract

A method of detecting a carbon nanotube, is carried out by: (a) reacting the carbon nanotube with a first transition metal complex that oxidizes the carbon nanotube in a first oxidation-reduction reaction, regenerating the reduced form of the first transition metal complex in a catalytic cycle; and (b) detecting the carbon nanotube by detecting the first oxidation-reduction reaction. The method may be used to detect a target compound (e.g., a protein, peptide or nucleic acid) directly or indirectly coupled to the carbon nanotube. Suitable transition metal complexes include but are not limited to Ru, Fe, Os or Re complexes.

Description

ELECTROCATALYTIC DETECTION OF CARBON NANOTUBES

H. Holden Thorp and Mary E. Napier

Field of the Invention

The present invention concerns methods for the electrocatalytic detection of carbon nanotubes, which methods provide a general system useful for the detection of molecules or binding events in which the carbon nanotubes serve as an easily detectable label.

Background of the Invention

The detection of individual DNA sequences in heterogenous samples of DNA provides a basis for identifying genes, DNA profiling, and novel approaches to DNA sequencing. One approach to DNA hybridization detection involves the use of surface bound DNA sequences which can be assayed using an analytical response that indicates hybridization of the surface-bound oligomer to a sequence in the heterogeneous sample. These prior analytical methods generally involve laser-induced fluorescence arising from a covalently attached label on the target DNA strand, which methods are not sensitive to single-base mismatches in the surface-bound duplex. For example, U.S. Pat. Nos. 5,143,854 and 5,405,783 to Pirrung et al.; Fodor, et al., Nature 364:555 (1993); Bains, Angew. Chem. 107:356 (1995); and Noble, Analytical Chemistry 67(5):201A (1995) propose surfaces or "chips" for this application. In an alternate method, proposed by Hall, et al., Biochem. and Molec. Bio. Inter. 32(1):21 (1994), DNA hybridization is detected by an electrochemical method including observing the redox behavior of a single stranded DNA as compared to a double stranded DNA. This technique is also not sensitive to single-base mismatches in the DNA sample. U.S. Patent Nos. 5,871,918 and 6,132,971 to Thorp et al., US Patent

Application No. 2003/0152960 to Thorp et al., and PCT Application WO2004/092708 to Thorp et al. describe methods and apparatus for electrically detecting a target molecule by detecting a preselected base in an oxidation-reduction reaction. The methods and apparatus disclosed therein may be used in a variety of applications, including DNA sequencing, diagnostic assays, and quantitative analysis. The methods can advantageously be implemented in a variety of different assay formats and structures, including multi-well plates, with a different assay carried out in each well. However, it would be useful to provide additional means for detecting oxidation- reduction reactions that can be detected in like manner.

Summary of the Invention

A first aspect of the invention is a method of detecting a carbon nanotube, comprising:

(a) reacting the carbon nanotube with a first transition metal complex that oxidizes the carbon nanotube in a first oxidation-reduction reaction (typically and preferably regenerating the reduced form of the first transition metal complex in a catalytic cycle); and (b) detecting the carbon nanotube by detecting the first oxidation-reduction reaction.

A second aspect of the invention is a method of detecting a target compound (e.g., a protein, peptide or nucleic acid), comprising:

(a) coupling (covalently or noncovalently) the target compound to a carbon nanotube;

(b) reacting the carbon nanotube with a first transition metal complex that oxidizes the carbon nanotube in a first oxidation-reduction reaction, regenerating the reduced form of the first transition metal complex in a catalytic cycle; and

(c) detecting the target compound by detecting the first oxidation-reduction reaction.

In some embodiments the target compound is a first member of a binding pair, the carbon nanotube further comprises a second member of a specific binding pair conjugated thereto, and the coupling step is carried out by specifically binding first and second members of the binding pair. The carbon nanotube can be dispersed in a solution such as an aqueous solution. In some embodiments carbon nanotube is complexed with a nucleic acid (e.g., a single stranded DNA, double stranded DNA, RNA or PNA). hi some embodiments the carbon nanotube can be coupled to a solid support (e.g., a solid support that comprises an electrode and a nonconductive layer, with the carbon nanotυbe coupled to the nonconductive layer).

Suitable transition metal complexes include but are not limited to Ru, Fe, Os or Re complexes, such as Ru(bpy)₃ ²⁺, Ru(Me₂ -bpy)₃ ²⁺, Ru(Me₂ -phen)₃ ²⁺, Fe(bpy)₃ ²⁺, Fe(5-Cl-phen)₃ ²⁺, Os(5-Cl-phen)₃ ²⁺, and ReO₂ (ρy)₄ ¹⁺.

The foregoing and other objects and aspects of the present invention are explained in greater detail in the drawings herein and the specification set forth below.

Brief Description of the Drawings Figure 1-3 schematically illustrate methods and apparatus of the invention of the present invention (e.g., a conductive substrate having a non-conductive layer formed thereon, with a CNT coupled to or deposited upon the non-conductive layer).

Figure 4. Electrochemical response for dT₆₀-wrapped CNT (dashed lines) and dT₆₀ oligonucleotide (solid lines) in solution with either 25 μM Ru(bpy)₃ ²⁺ or with 25 μM Fe(dmb)₃ ²⁺ (inset). The CNT concentration in these samples is 0.01 mg/mL and an oligonucleotide concentration of 0.018 mg/mL (1 μM). The scan rate is 25 mV/s. The current enhancement observed for the dT₆₀ wrapped-CNT is due to oxidation of the CNT by electrogenerated Ru(III). No current enhancement was observed for the dT₆o wrapped CNT when Fe(dmb)₃ ²⁺ is the metal mediator at this concentration and the time scale of this experiment.

Figure 5. A) Ratio of the CA traces obtained in the presence of CNT (i_cat) and in the absence of CNT (i_d) as a function of the square root of the time. The CA plots were generated using low salt conditions. The entire time trace is shown for 0.02 mg/mL CNT, (upper line), 0.01 mg/mL CNT (middle line) and 0.005 mg/mL CNT (lower line). The solid lines are the linear fits to the early, fast phase and the later, slower phase. The slope of the lines fit to the data is equal to (πk'C_z*)^1/2. B) Cyclic voltammograms of dT₆₀-wrapped CNT with 25 μM Ru(bpy)₃ ²⁺ showing the experimental and simulations. All of the simulations are in solid lines and the experimental are as follows; 0.005 mg/mL CNT (dash), 0.01 mg/mL CNT (dot) and 0.02 mg/mL CNT (dot-dash).

Figure 6. Cyclic voltammogram of dT₆₀-wrapped CNT at 0.01 mg/mL CNT concentration with 25 μM Ru(bpy)₃ ²⁺. Experimental data represented by dashed lines and the simulated data with solid lines. Only the first four C reaction steps were used to fit the data.

Figure 7. Electrochemical response for A) d(T)₆₀, B) d(T)₅₀(G)i₀ and C) d(GT)₃₀-wrapped CNT in solution with 25 μM Ru(bpy)₃ ²⁺ (red solid) at three CNT concentrations 0.005 (black dash line), 0.01 (blue dot line) and 0.02 mg/mL (green dot-dash line). See Table 4 for guanine concentration. The scan rate is 25 mV/s. The current enhancement observed for the DNA-wrapped CNT is due to oxidation of both the guanine base and the CNT by electrogenerated Ru(III).

Figure 8. Electrochemical response for d(T)₆o-wrapped CNT (blue diamond), d(T)₅o(G) _lo- wrapped CNT (black square) and d(GT)₃o-wrapped CNT (green x) at a CNT concentration of 0.02 mg/mL. As the guanine concentration of the wrapping sequence increases the current also increases for the same CNT concentration. Also shown is the electrochemical response for each oligonucleotide sequence, d(T)_{60 (}blue triangle), d(T)₅o(G)io (black cross) and d(GT)₃₀ (green circle), in the absence of CNT at the same concentration as that present in the CNT containing solutions (Table 4).

Figure 9. Cyclic voltammograms of A) d(T)₆o-wrapped CNT, B) d(T)₅₀(G)i₀- wrapped CNT, and C) d(GT)₃₀-wraρped CNT with 25 μM Ru(bpy)₃ ²⁺ in 0.1 M sodium phosphate buffer, pH 7, showing the experimental data and simulations. All of the simulations are red solid lines and the experimental data are shown as follows: 0.005 mg/mL CNT (black dash line), 0.01 mg/mL CNT (blue dot line) and 0.02 mg/mL CNT (green dot dash line).

Figure 10. Electrochemical response for A) 0.01 mg/mL d(T)₆₀-wrapped CNT and d(T)₆o oligonucleotide in 25 μM Ru(bpy)₃ ²⁺ and 0.1 M sodium phosphate solution, pH 7 in the presence and absence of 0.8 M NaCl. B) 0.01 mg/mL d(GT)₃₀- wrapped CNT and d(GT)₃₀ oligonucleotide in 25 μM Ru(bpy)₃ ²⁺ and 0.1 M sodium phosphate solution, pH 7 in the presence and absence of 0.8 M NaCl. The black solid trace is 25 μM Ru(bpy)₃ ²⁺ in both the presence and absence of 0.8 M NaCl. The blue traces are DNA-wrapped CNT (dot dash blue) and oligonucleotide alone (long dash blue) low salt buffer. The red traces are the DNA-wrapped CNT (dash red) and oligonucleotide alone (red dot) in high salt buffer. The d(T)₍,o oligonucleotide in the absence of CNT yielded nearly identical CV traces in both the low and high salt buffers. The scan rate is 25 mV/s. Detailed Description of the Preferred Embodiments

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

"carbon nanotube" refers to a generally hollow article composed primarily of carbon atoms. The carbon nanotube can be doped with other elements, e.g., metals. "Nucleic acid" as used herein includes a polymer of RNA, DNA, or peptide nucleic acid (PNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. A nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA. "Peptide nucleic acid" refers to a material having stretches of nucleic acid polymers linked together by peptide linkers.

"Nanotube-nucleic acid complex" means a composition comprising a carbon nanotube associated with at least one nucleic acid molecule. Typically the association between the nucleic acid and the nanotube is by van der Waals bonds or some other non-covalent means.

"Binding pair" refers to chemical or biopolymer based couples that bind specifically to each other. Common examples of binding pairs are immune-type binding pairs, such as antigen/antibody or hapten/anti-hapten systems. Suitable binding pairs include, but are not limited to, glutathione-S-transferase/glutathione, όxhistidine Tag/Ni-NTA, streptavidin/biotin, S-protein/S-peptide, cutinase/phosphonate inhibitor, antigen/antibody, hapten/anti-hapten, folic acid/folate binding protein, and protein A or G/immunoglobulins. Another example of a binding pair is a negatively charged phosphate backbone of a nucleic acid molecule, with the second member being a positively charged surface. Still other binding pairs are nucleic acids and complementary nucleic acids that hybridize to one another.

"Solid support" means a material suitable for the immobilization of a nanotube-nucleic acid complex. Typically the solid support provides an attachment of a member of a binding pair through which the complex is captured and immobilized. The solid support may include one or more electrodes along with a nonconductive portion or layer, with the binding pair coupled to or immobilized on the nonconductive layer.

The disclosures of all United States patent references cited herein are to be incorporated by reference herein in their entirety.

1. Carbon nanotubes.

Carbon nanotubes useful for carrying out the present invention are known. Examples include but are not limited to those described in US Patent Nos. 6,921,575; 6,914,381; 6,911,260; 5,872,236; 6,827,918; 6,821,730; and 5,543,378.

Carbon nanotubes used to carry out the invention may be from about 0.5-2 nm in diameter where the ratio of the length dimension to the narrow dimension, i.e., the aspect ratio, is at least 5. In general, the aspect ratio is between 10 and 2000. Carbon nanotubes are comprised primarily of carbon atoms, however they may be doped with other elements, e.g., metals. Carbon nanotubes used in the invention can be either multi-walled nanotubes (MWNTs) or single-walled nanotubes (SWNTs). A MWNT, for example, includes several concentric nanotubes each having a different diameter. Thus, the smallest diameter tube is encapsulated by a larger diameter tube, which in turn, is encapsulated by another larger diameter nanotube. A SWNT, on the other hand, includes only one nanotube. See, e.g., US Patent Application 20040132072.

Carbon nanotubes (CNT) may be produced by a variety of methods, and are additionally commercially available. Methods of CNT synthesis include laser vaporization of graphite (A. Thess et al. Science 273, 483 (1996)), arc discharge (C. Journet et al., Nature 388, 756 (1997)) and HiPCo (high pressure carbon monoxide) process (P. Nikolaev et al. Chem. Phys. Lett. 313, 91-97 (1999)). Chemical vapor deposition (CVD) can also be used in producing carbon nanotubes (J. Kong et al. Chem. Phys. Lett. 292, 567-574 (1998); J. Kong et al. Nature 395, 878-879 (1998); A. Cassell et al. J. Phys. Chem. 103, 6484-6492 (1999); H. Dai et al. J. Phys. Chem. 103, 11246-1 1255 (1999)).

Additionally CNTs may be grown via catalytic processes both in solution and on solid substrates (Yan Li, et al., Chem. Mater.; 2001; 13(3); 1008-1014); (N. Franklin and H. Dai Adv. Mater. 12, 890 (2000); A. Cassell et al. J. Am. Chem. Soc. 121, 7975-7976 (1999)).

Carbon nanotubes may be dispersed in an aqueous solution by complexing them with a nucleic acid as described in US Patent Application No. 20040132072 to Zheng and Diner. The nucleic acid used to form the nucleic acid may serve as a ligand or member of a specific binding pair for coupling a target molecule thereto, or a different ligand or member of a specific binding pair may be coupled to the nucleic acid as also described therein.

In certain embodiments, carbon nanotubes may be derivatized with reactive groups to facilitate attachment to ligands, analytes, probes or the like. In preferred embodiments, each nanotube may be derivatized to contain a single reactive group at one end of the tube, although it is contemplated that nanotubes may contain more than one reactive group located anywhere on the tube. In a non-limiting example, nanotubes may be derivatized to contain carboxylic acid groups (U.S. Pat. No. 6,187,823). Carboxylate derivatized nanotubes may be attached to nucleic acid probes or other analytes by standard chemistries, for example by carbodiimide mediated formation of an amide linkage with a primary or secondary amine group located on a probe or analyte. The methods of derivatization and cross-linking are not limiting and any reactive group or cross-linking methods known in the art may be used. See, e.g., US Patent No. 6,821 ,730.

2. Transition metal complexes.

Transition metal complexes (also referred to as "mediators" herein) that enable or make possible electron transfer to a CNT as described above are described in, for example, U.S. Patent Nos. 5,871,918 and 6,132,971 to Thorp et al. An example is Ru(bpy)₃ ²⁺. Other examples include, but are not limited to, Ruthenium²⁺(2,2'- bipyridine)₃ ("Ru(bpy)₃ ²⁺"); Ruthenium²⁺(4,4'-dimethyl-2,2'-bipyridine)₃ ("Ru(Me₂- ^bPy)3²⁺"); Ruthenium²⁺(5,6-dimethyl-l,10-phenanthroline)₃ ("Ru(Me₂-phen)₃ ²⁺"); Iron²⁺(2,2'-bipyridine)₃ ("Fe(bpy)₃ ²⁺"); Iron²⁺(4,4'-dimethyl-2,2'-bipyridine)₃ ("Fe(Me₂-bpy)₃ ²⁺"); W⁺(5-chlorophenanthroline)₃ ("Fe(5-Cl-phen)₃ ^2+M);

Iron²⁺(4,4'-dimethyl-2,2'-bipyridine)(bipyridine)₂ ("Fe(Me₂-bpy)(bpy)₂ ²⁺");

Iron²⁺(4,4'-dimethyl-2,2'-biρyridine)₂(bipyridine) ("Fe(Me₂-bpy)₂(bpy)²⁺");

Osmium²⁺(2,2'-bipyridine)₃ ("Os(bpy)₃ ²⁺"); Osmium²⁺(4,4'-dimethyl-2,2'- bipyridine)₃ ("Os(Me₂-bpy)₃ ²⁺"); Osmium²⁺(5-chlorophenanthroline)₃ ("Os(5-Cl- phen)₃ ²⁺"); Osmium²⁺(4,4'-dimethyl-2,2'-bipyridine)(bipyridine)₂ ("Os(Me₂- bpy)(bρy)₂ ²⁺"); Osmium²⁺(4,4'-dimethyl-2,2'-bipyridine)₂(bipyridine) ("Os(Me₂- bpy)₂(bpy)²⁺"); dioxorhenium¹⁺phosphine; and dioxorhenium^I+pyridine ("ReO₂(py)₄ ¹⁺"). Some anionic complexes useful as mediators are: Ru(bpy)((SO₃)₂- bpy)₂ ^" and Ru(bpy)((CO₂)₂-bpy)₂ ^" and some zwitterionic complexes useful as mediators are Ru(bpy)₂((SO₃)₂-bpy) and Ru(bpy)₂((CO₂)₂-bρy) where (SO^-bpy^2" is 4,4'-disulfonato-2,2'-bipyridine and (CO₂)₂-bpy^2" is 4,4'-dicarboxy-2,2'-bipyridine. Derivatives of the ferrocene molecule may also be used. Suitable substituted derivatives of the pyridine, bipyridine 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; 5,5'-dimethyl-2,2'-bipyridine; 6,6'-dimethyl-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',4"-triamino-2,2',2"-teφyridine; 4,4',4"-triethylenediamine-2,2',2"- teφyridine; 4,4',4"-trihydroxy-2,2',2"-terpyridine; 4,4',4"-trinitro-2,2',2"-teφyridine; 4,4',4"-triphenyl-2,2',2"-teφyridine; 4,7-diamino-l,10-phenanthroline; 3,8-diamino- 1 , 10-phenanthroline; 4,7-diethylenediamine- 1 , 10-phenanthroline; 3,8- diethylenediamine- 1 , 10-phenanthroline; 4,7-dihydroxyl- 1 , 10-phenanthroline; 3,8- dihydroxyl- 1 , 10-phenanthroline; 4,7-dinitro- 1 , 10-phenanthroline; 3,8-dinitro- 1,10- phenanthroline; 4,7-diρhenyl- 1 , 10-phenanthroline; 3,8-diphenyl- 1 , 10-phenanthroline; 4,7-disperamine- 1 , 10-phenanthroline; 3 ,8-disperamine- 1 , 10-phenanthroline; dipyrido[3,2-a:2',2'-c]phenazine; 4,4'-dichloro-2,2 '-bipyridine; 5,5'-dichloro-2,2'- bipyridine; and 6,6'-dichloro-2,2'-bipyridine.

3. Assay formats. The present invention is useful for the detection of CNTs per se, for example when it is desired to test the products of systems or methods for the synthesis, derivitization, isolation, and/or purification of CNTs. The present invention is useful for the detection of target compounds, with the CNT serving as an electrochemical label or electrochemical detectable group that can be detected by the methods described herein.

The present invention can be used to detect a member of a binding pair. Binding pairs are any two molecules that have a high affinity specific interaction between the two members (herein often referred to as a "first member" and a "second member" or vice versa). Numerous binding pairs are known. See, e.g., US Patent

Nos. 6,921,638; 6,649,138; 6,551,843; 6,306,610; and 6,261,554. Examples of binding pairs include but are not limited to: nucleic acid and a corresponding nucleic acid that hybridizes thereto; antibody (including fragments or derivatives and both polyclonal and monoclonal) and the corresponding antigen; receptors and their ligands (including proteinaceous ligands and nonprotein ligands such as steroids, drugs, etc.); well known binding pairs such as avidin/biotin or streptavidin/biotin; peptide structures that specifically interact in solution and the like. Either member of the binding pair may be coupled to the CNT in accordance with known techniques and the CNT used as a detectable label in the methods described herein to detect binding of the two compounds, which binding may be carried out by any suitable assay format in accordance with known techniques.

The present invention can be carried out with any suitable assay format, including but not limited to those described in US Patent No. 5,871,918 to Thorp et al., US Patent No. 6,132,971 to Thorp et al., US Patent Application No. 2003/0152960 to Thorp et al., and PCT Application WO 2004/092708 to Thorp et al.

The present invention can be carried utilizing the assay formats described in US Patent Application No. 2004/0132072 to Zheng et al., combined with the detection techniques described herein.

For example, the CNT can be coupled to an antibody, and the antibody utilized to detect an antigen or binding partner thereto (with the CNT as a label or detectable group) in any suitable assay such as a homogeneous assay or a sandwich assay {e.g., by providing a second antibody immobilized on a solid support as described herein).

In another example, the CNT can be complexed with a nucleic acid and the nucleic acid used as a probe (with the CNT as a label or detectable group) to bind to and detect another nucleic acid. In still another example, the CNT can be coupled to a peptide or nucleic acid, and the peptide or nucleic acid utilized as a probe (with the CNT as a label or detectable group) to bind to and detect another protein, peptide or nucleic acid.

Where the target molecule is a nucleic acid the target molecule can be amplified prior to reaction and detection. Amplification may be carried out by any suitable means. See generally D. Kwoh and T. Kwoh, Am. Biotechnol. Lab. 8, 14-25 (1990). Examples of suitable amplification techniques include, but are not limited to, polymerase chain reaction (including, for RNA amplification, reverse-transcriptase polymerase chain reaction), ligase chain reaction, strand displacement amplification, transcription-based amplification (see D. Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173-1177 (1989)), self-sustained sequence replication (or "3SR") (see J. Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874-1878 (1990)), the Q.beta. replicase system (see P. Lizardi et al., Biotechnology 6, 1197-1202 (1988)), nucleic acid sequence- based amplification (or "NASBA") (see R. Lewis, Genetic Engineering News 12 (9), 1 (1992)), the repair chain reaction (or "RCR") (see R. Lewis, supra), and boomerang DNA amplification (or "BDA") (see R. Lewis, supra). The bases incorporated into the amplification product may be natural or modified bases (modified before or after amplification), and the bases may be selected to optimize subsequent electrochemical detection steps. Techniques for amplification are known and described in, among other things, U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188; G. Walker et al., Proc. Natl. Acad. Sci. USA 89, 392-396 (1992); G. Walker et al., Nucleic Acids Res. 20, 1691-1696 (1992); R. Weiss, Science 254, 1292 (1991).

In one embodiment amplification is carried out by asymmetric polymerase chain reaction, in accordance with known techniques or variations thereof which will be apparent to those skilled in the art. See, e.g., Loh et al., Science, 243 (4888): 217- 220 (Jan 13 1989)(PCR with primer attached to a surface); U.S. Patent No. 5,075,216; U.S. Patent No. 6,391,546.

4. Detection of reactions. The transition metal complexes are reacted with CNTs (serving as labels or detectable groups) under conditions sufficient to effect the oxidation-reduction reaction of the transition metal complex with the CNT in a catalytic reaction. The solution in which the oxidation-reduction reaction takes place may be any suitable solution for solubilizing the components of the assay and preferably comprises water. Suitable conditions for permitting the oxidation-reduction reaction to occur will be known to those skilled in the art.

The occurrence of the oxidation-reduction reaction of the invention may be detected according to any suitable means known to those skilled in the art. For example, the occurrence of the oxidation-reduction reaction may be detected using a detection (working) electrode to observe a change in the electrochemical signal, which is indicative of the occurrence of the oxidation-reduction reaction. An electrode suitable for the detection of labels in accordance with the methods described herein comprises a conductive substrate having a working surface thereon, and is sensitive to the transfer of electrons between the mediator and the label.

Generally, a reference electrode and an auxiliary electrode are also placed in contact with the mediator solution in conjunction with the detection electrode. Suitable reference electrodes are known in the art and include, for example, silver/silver chloride (Ag/ AgCl) electrodes, saturated calomel electrodes (SCE), and silver pseudo reference electrodes. A suitable auxiliary electrode is a platinum electrode.

The detection of the electrochemical signal produced by the catalytic oxidation-reduction of labels permits the determination of the presence or absence of specific substances in a sample. As used herein, terms such as determining or detecting "the presence or absence" of a substance as used to describe the instant invention, also include quantitation of the amount of the substance. In the invention, the transition metal mediator is oxidized by an electrode. Then, the mediator is reduced by the label and then reoxidized at the electrode. Thus, there is electron transfer from the label to the transition metal mediator resulting in regeneration of the reduced form of the transition metal mediator as part of a catalytic cycle. The step of determining the presence or absence of target in a sample typically includes: (i) measuring the electrochemical signal generated by the oxidation-reduction reaction of the mediator at electrodes that are and are not capable of specifically binding the target, (ii) comparing the measured signal from the transition metal complex at both electrodes, and then (iii) determining whether or not the electrochemical signal generated from the mediator at the electrode that is capable of binding the target is essentially the same as, greater than, or less than, the electrochemical signal generated from the mediator at the electrode that does not bind the target. The step of measuring the electrochemical signal may be carried out by any suitable means. For example, the difference in electrochemical signal may be determined by comparing the electrochemical signal (such as current or charge) from electrodes which are and are not capable of binding the target at the same scan rate, mediator concentration, buffer condition, temperature, and/or electrochemical method.

The electrochemical signal associated with the oxidation-reduction reaction may be measured by providing a suitable apparatus in electronic communication with the detection electrode. A suitable apparatus is a potentiostat capable of measuring the electronic signal that is generated so as to provide an indication of whether or not a reaction has occurred between the label and the mediator. The electronic signal may be characteristic of any electrochemical method, including cyclic voltammetry, normal pulse voltammetry, chronoamperometry, and square-wave voltammetry, with chronoamperometry and cyclic voltammetry being the currently preferred forms. In cyclic voltammetry, the potential of the electrochemical system is varied linearly from an initial potential between 0-800 mV to a final potential between 500- 1600 mV at a constant scan rate (0.01 mV/s to 200 V/s). When the final potential is reached, the scan direction is reversed and the same potential range is swept again in the opposite direction. The preferred scan rate for Ru(bpy)₃ ²⁺ is 1 -20 V/s (for surface confined species) or 5m V/s- 1 V/s (when in solution) with a 0 mV initial potential and a 1400 mV final potential. The current is collected at each potential and the data is plotted as a current versus potential scan. For lower-potential mediators, such as Os(bpy)₃ ²⁺ and Os(Me₂-bpy)₃ ²⁺, instead of scanning from between 0-800 mV to between 500-1600 mV, it is preferable to scan from about between 0-100 raV to between 300-1000 mV (vs. a Ag/AgCl reference electrode) because of the lower redox potentials required to oxidize these mediators.

In chronoamperometry as used in the invention herein, the electrochemical system is stepped from an initial potential between 0 mV-800 mV directly to a final potential between 500-1600 mV and held there for some specified period of time (50 μs to 10 s) and the current is collected as a function of time. If desired, 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 for Ru(bpy)₃ ²⁺ is from between 0-800 mV to 1300 mV (vs. Ag/AgCl) with a collection time of from 50-1000 ms. For lower potential mediators, such as Os(bpy)₃ ²⁺ and Os(Me₂-bpy)₃ ²⁺, it is preferable to step from about 0-100 mV to 300-1000 raV (vs. Ag/AgCl).

In chronocoulometry, a potential step is also applied. For use in the invention herein, starting at the initial potential (0 mV-800 mV), the electrochemical system is stepped directly to the final potential (500 mV-1600 mV) (vs. Ag/ AgCl). The electrochemical system is held at the final potential for some specified period of time (50 μs to 10 s) and the charge is collected as a function of time. If desired, 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. Figures 1-3 represent various non-limiting embodiments of the methods and apparatus of the invention. The steps represented in Figure 2 are as follows: In step 1 , the surface of a substrate is modified in accordance with known techniques to immobilize or bind a capture probe thereto. The DNA wrapped around the CNT has a portion which is complementary to the capture probe on the surface. Step 2 shows is the hybridization of the nucleic acid- wrapped carbon nanotube (If the DNA wrapped around the CNT is G free then you are only detecting electrons the CNTs brought to the surface through the hybridization event. DNA sensor or CNT sensor. If the DNA wrapped around the CNT is G rich then you are seeing electrons from both the DNA and CNT. DNA sensor). Step 3 shows the hybridization of a second target nucleic acid. Step three is included because of the desireability of dispersing or solubilizing the CNT in an aqueous solution and the difficulty of doing so in the field. Note that step 3 is one possible embodiment of a nucleic acid biosensor, and step 3 represents the preparation of a biosensor only if a guanine free-DNA is used (or a second representation of a biosensor if the wrapping DNA itself is the DNA to be detected). The present invention is explained in greater detail in the following non- limiting Examples.

Example 1-3

Methods Example 1

Carbon Nanotube Dispersion and Purification

Reagents. Water was deionized using a MiIIiQ Plus water purifications system. Synthetic oligonucleotides were obtained from MWG Biotech (High Point, NC) as lyophilized solids, brought up in filtered, deionized water and the concentration measured spectrophotometrically using a Cary 300 Bio UV-Vis spectrophotometer. CoMoCAT nanotubes were purchased from Southwest Technologies, Norman, OK. All other chemicals used were purchased from Sigma Aldrich (St. Louis, MO).

Carbon nanotube dispersion and purification. The oligonucleotide sequences used to suspend the carbon nanotube are given in Table 1. Oligonucleotide concentrations were determined spectrophotometrically per strand.

Table 1. Oli onucleotide se uences used to sus end the carbon nanotubes

DNA dispersion of the CNT was accomplished using a similar procedure to the one published by the Dupont researchers.^1"3 The as produced CoMoCAT process SWeNT™ purified single walled carbon nanotube (SWCNT) manufactured by Southwest Technologies (Norman, OK) were used. The CoMoCAT process developed by Resasco produces a narrow (n,m) distribution of nanotubes.⁴ The CNTs were mixed with oligonucleotide in 0.1 M sodium phosphate buffer and sonicated (Sonics, VC 130 PB) in an ice water bath for two hours at 3 W. The mixture was then centrifuged for 90 minutes at 16,000 g to remove the unsuspended carbon nanotubes. The suspended carbon nanotube solution was sonicated again for an additional 60 minutes at 3W. The mixture was again centrifuged for an additional 60 minutes and the suspended carbon nanotubes were separated from the unsuspended carbon nanotubes. The mixture was them filtered through a 0.45 micron Millex-HV 4 mm filter. A substantial fraction of the oligonucleotide in solution was still free in solution, not wrapped around a carbon nanotube. A procedure was developed to separate the free DNA from the DNA-wrapped CNT using YMlOO molecular weight cutoff filters. No more than 300 μL of the DNA-wrapped CNT solution was placed on the YMlOO filter and the filter centrifuged at 10,000 g for 10 minutes. Free DNA, oligonucleotide not associated with a CNT, passes through the filter and the DNA- wrapped CNT remains on the filter. Approximately 75 μL of 0.1 M phosphate buffer at pH 7 was added to the filter, and the filter was then turned upside down in a fresh tube and centrifuged at 4,000 g'for 4 minutes to transfer the DNA-wrapped CNT. If the DNA-wrapped CNT remained on the YMlOO filter, an additional 50-75 μL of sodium phosphate buffer was added and the filter was centrifuged again. To obtain DNA to CNT (mg/mL) ratios of approximately 2.5:1, required at least two passes through the YMlOO filter. The samples were fully characterized before application to the YMlOO filters and following each pass through the filter, using optical absorption spectroscopy on a Shimadzu UV- 1601. Diner and Zheng report that 1 OD_99Onn, is equivalent to approximately 13 μg carbon nanotube material/mL assuming a 1:1 mass ratio between the CNT and DNA derived from computer modeling.¹ The background subtracted absorption at 990 nm was used to calculate the CNT concentration. The oligonucleotide concentrations were calculated using the optical absorption spectra collected using a Cary 300Bio UV-Vis spectrophotometer and the appropriate extinction coefficient. Calculating the DNA concentration for the DNA-wrapped CNT is complicated by a rising background and a broad adsorption peak at 270 nm resulting from graphitic impurities suspended in solution.⁵ The background subtracted absorption at 260 nm was used to calculate the oligonucleotide concentration.

Example 2

Electrochemical Methods

Electrochemistry. All electrochemical experiments were performed using a CH Instruments 600 series potentiostat. Electrochemical measurements were performed in cells described previously.⁶ The working electrode was an indium tin oxide (ITO) electrode (working area approximately 0.32 cm²) from Applied Films Coφoration (Longmont, CO). The Ag/AgCl reference electrode was purchased from Cypress Systems, Inc (Lawrence, KS). ITO electrodes were cleaned by sonicating in MiIH-Q water for 15 minutes, 2-propanol for 15 minutes, and Milli-Q water twice, 15 minutes each. After cleaning the electrodes were allowed to air dry prior to use. The potential was applied via a platinum wire counter electrode. The cyclic voltammetry (CV) data was collected using the following parameters, potential range 0-1.3 V, scan rate 25 mV/s and a metal mediator concentration of 25 μM. A CV of the buffer without metal complex was subtracted from the voltammograms for samples without suspended CNTs. A CV of the DNA-wrapped CNT sample without metal complex was subtracted from the voltammograms for samples with suspended CNTs. The chronoamperometry data was collected using the following parameters, potential step from 0.7 V to 1.2 V (similar results were obtained for steps to 1.25 V) and a pulse width of 0.5 s. The CA data was not background subtracted.

A 0.1 M sodium phosphate buffer, pH 7, is used for all experiments except where a change is specifically noted.

Example 3 Digital Simulation of Cyclic Voltammograms Digital Simulation. The digital simulations were performed using the

DigiSim software package (Bioanalytical Systems, West Lafayette, IN).⁷ The following parameters were used in every simulation; electrode area 0.32cm², scan rate (v) 25mV/s, E₀=I.06 V vs Ag/AgCl (for Rubpy₃ ²⁺). The diffusion coefficients used are 6.0 x 10^"6 cm²/s for Rubpy₃ ²⁺ and 1.0 x 10^"7 cm²/s for carbon nanotubes (CNT). The diffusion coefficient for CNT is an estimate based on the diffusion coefficient calculated assuming a rigid rod behavior.⁸'⁹ The concentration of Rubpy₃ ²⁺ in all simulations is 25μM. The concentration of CNT will be indicated in the simulation figure caption. The homogeneous electron transfer rate constants (k_f) were determined by fitting the cyclic voltammogram. Examples 4-10 Results

Example 4

Electrocatalytic Oxidation of d(T)₆₀-Wrapped CNT

Shown in Figure 4 are the cyclic voltammograms of 25 μM Ru(bpy)₃ ²⁺ and 25 μM Fe(dmb)₃ ²⁺ (inset) with either 1 μM dT₆o oligonucleotide or dT_όo-wrapped CNT at a concentration of 1 μM dT₆o and 0.01 mg/mL CNT. Because the dT₆₀ oligonucleotide contains no oxidizable guanines there is no current enhancement for the samples that contain the metal complex and the oligonucleotide compared to cyclic voltammograms of the metal mediator alone (not shown). However, addition of the dTόo-wrapped CNT produces dramatic current enhancements even when the metal complex catalyst is in a 25-fold excess compared to the oligonucleotide. The current enhancement in the presence of dT₆₀-wrapped CNT is due to oxidation of the CNT by electrogenerated Ru(III) and subsequent recycling of the metal complex redox reaction. No current enhancement was observed for the dT₆o-wrapped CNT with Fe(dmb)₃ ²⁺, suggesting that the rate of electron transfer is significantly reduced at this lower potential. At higher CNT concentrations, detectable catalytic current could be observed for Fe(dmb)₃ ²⁺, consistent with the potentials estimated from the redox titration experiment performed by Zheng and Diner.¹ It is very important to note that no oxidation current was observed for the wrapped CNT in the absence of metal complex, which is consistent with our observations on other oxidizable polyanions.¹⁰ To our knowledge, this is the first observation of electrocatalytic oxidation of a CNT in fluid solution. As with DNA, relatively low quantities of CNT are required to obtain this signal; however, the absolute amount of wrapped-CNT needed to induce current enhancement is much smaller than for DNA, even for sequences containing many guanine bases. The ITO-Ru(bpy)₃ ²⁺ system provides a convenient platform for studying electrocatalytic oxidation of DNA-solubilized CNTs, which are surprisingly effective electron donors. Example 5 Analysis of the Chronoamperometric Response of d(T)₆o-Wrapped CNT

Figure 5 A shows the chronoamperometric (CA) response for Ru(bpy)₃ ²⁺- mediated oxidation of dT₆o-wrapped CNT in 0.1 M sodium phosphate buffer at pH 7. The plot of the current in the presence of CNT (i_cat) divided by the response in the absence of CNT (i_d) versus t^1/2 for three different concentrations of carbon nanotubes shows a biphasic response. For each curve there are two linear regimes, an early, fast regime and a slower regime at longer times. Similar responses have been previously observed with guanine-containing DNA, and the two regimes were interpreted as two phases of the electrocatalytic reaction." The apparent second-order rate constants were determined from the slope of the best fit lines at the early and late times with slopes equal to (πk'C_z ^*)^1/2.¹² The rate constants (k') obtained were calculated using a concentration of reducing equivalents (C_z ^*) derived from estimates by Zheng and Diner of an average CNT length of 140 nm and a number of redox-active sites equal to one per 5 ran.^1"3 The rate constants obtained from the best fits to the data do not exhibit any systematic dependence on the carbon nanotube concentration, as expected. The fast phase exhibits an average rate constant of 6x10⁶ M^-1S ¹ and the slow phase exhibits an average rate constant of 3.5x10⁴ M^-1S^"1; note again that these rate constants are expressed in terms of 5 nm reducing equivalents not for the whole CNT. The rate constants obtained are reported in Table 2 and were calculated using a concentration which is equivalent to the active site concentration of the carbon nanotube.

Table 2: Second-Order Rate Constants for the Oxidation of d(T)₆o-wrapped NTs by Ru(bpy)₃ ²⁺ Generated by CA

The active site concentration is defined by the estimate of Ie^" for every 5 nm length of carbon nanotube calculated by Diner et al. from the redox titration experiments.¹ The active site concentration calculations assume an average length of 140 nm for each carbon nanotube.

Example 6

Analysis of Cyfclic Voltammograms of d(T)₆o-Wrapped CNT The rate constants derived from the chronoamperometric responses could be used to develop digital simulations of the cyclic voltammetry collected for the same CNT concentrations. The experimental data and the simulated curves for the Ru(bpy)₃ ²⁺-mediated CNT oxidation are shown in Figure 5B. To simulate the electrocatalytic signal, an EC mechanism in the following form was employed:

Ru(bpy)₃ ²⁺ -> Ru(bpy)₃ ³⁺ + e (E) Ru(bpy)₃ ³⁺ + CNT — ► Ru(bpy)₃ ²⁺ +CNT_0x (C)

In contrast to simulations of DNA oxidation, ten successive C reactions were required to account for the observed signal. The number of C reactions needed was determined systematically by successive testing of the addition of each new C reaction at various homogeneous rate constants until the simulated electrocatalytic signal was similar to the experimental signal. The rate constants for the first homogeneous reaction (C) determined by simulating the data ranged from 4 - 5 x 10⁶ M^-1S^"1, in excellent agreement with the rate constants derived from the fast portion of the CA trace. The rate constants that describe the later reaction steps are very similar to the rate constants derived from the slower portions of the CA trace.

The rate constants derived from the CA responses are described well by simulations of the CV data collected for the same carbon nanotube concentrations. The heterogeneous electron transfer rate constants (k_f) derived from the simulated data are given in Table 3. Ten C reactions were used in order to account for the observed signal. The later reactions are extremely important for achieving a good fit to the experimental data. Shown in Figure 6, is the experimental data for the 0.01 mg/mL CNT concentration and the simulated data in which only the first four C reaction steps were used to fit the data. This simulation is unable to account for all of the current observed. Clearly the later, slower reactions are contributing substantially to the current observed. To obtain the fits shown, all 10 C reactions were required. The presence of extensive follow-up chemistry is not surprising, since each 5 nm segment contains nearly 500 carbon atoms that can undergo oxidation in neutral aqueous solution with a continuous supply of electrogenerated Ru(III).

Table 3: Simulated Rate Constants at Three Carbon Nanotube Concentrations

The voltammetric response shown in Figure 4 appears similar to that expected for a pseudo-first order excess of reductant,¹² yet the CNT concentration is far below that of the Ru(bpy)₃ ²⁺ catalyst; note that the absolute CNT concentration in Figure 4 is 0.65 μM. This effect arises from the multi-electron nature of the oxidation, which arises both from multiple electron donor sites in the CNT¹ as well as the over- oxidation of each site (by ten electrons in these early simulations). The high absolute rate constants likely also point to a role for electronic derealization along the CNT; the fractions used here are mostly semiconducting with some metallic CNTs.¹ The convenience of the Ru(bpy)₃ ²⁺ -ITO system should allow for detailed study of these properties and a rigorous description of the CNT oxidation reaction.

Example 7

EIectrocatalytic Oxidation of DNA-Wrapped CNT: Inclusion of Guanine Base in the Wrapping Sequence

Sonication of carbon nanotubes in an aqueous solution containing single stranded oligonucleotide sequences efficiently disperses carbon nanotubes in the buffer solution. The electrochemical response for carbon nanotubes (CNT) wrapped in 60-mer oligonucleotide sequences containing either zero, one, ten or thirty guanine bases was recorded and compared to the electrochemical response for the oligonucleotide in solution with no suspended carbon nanotube present and to the electrochemical response for the metal mediator alone. Shown in Figure 7 are the cyclic voltammograms of 25 μM Ru(bpy)₃ ²⁺ and d(T)₆₀, d(T)₅₀(G)i₀ and d(GT)₃o- wrapped CNT at 0.005, 0.01 and 0.02 mg/mL CNT in 0.1 M sodium phosphate buffer, pH 7, recorded at 25 mV/s. Given in Table 4 are the sequence specific oligonucleotide concentrations measured in solution at each CNT concentration used in these experiments along with the corresponding guanine concentration. The cyclic voltammograms for the d(T)₅₉(G)i wrapping sequence which contain only one guanine are not shown, because the data set for the oligonucleotide sequence containing only one guanine is very similar to the data set for d(T)₆o. The signal contributed by the one guanine in the wrapping sequence does not significantly influence the electrochemical response.

Table 4: CNT and Corresponding Oligonucleotide Wrapping Sequence Concentration

Universally, the addition of the suspended CNT to a solution containing

Ru(bpy)₃ ²⁺ produced a significant current enhancement in the voltammograms when compared to voltammograms for solutions which were CNT-free or contained only the metal mediator. Similar results were recorded for electrochemical experiments on d(T)₃₀, d(T)₆o and d(T)i2₀-wrapped CNT. The universal increase in current with the inclusion of the carbon nanotube in solution is observed irrespective of the sequence used to wrap and suspend the CNT. As the number of guanines in the wrapping sequence increases, the current observed increases as well (Figure 7), suggesting that the signal collected is a result of mediated electron transfer from both the guanine base and the carbon nanotube to the metal mediator. Neither the guanine base nor carbon nanotubes undergo direct oxidation at 1.1 V, the oxidation potential for guanine and carbon nanotubes.

Shown in Figure 8 is the electrochemical response for d(T)₆₀, d(T)₅₀(G)₁₀ and d(GT)₃o-wrapped CNT at 0.02 mg/mL CNT, as well as the electrochemical response for each oligonucleotide only in solution with no CNT present. The guanine concentration in each solution is the same as that measured in the solution of the 0.02 mg/mL suspended CNTs. The oligonucleotide and guanine concentration are presented in Table 4. All of the solutions were prepared using 0.1 M sodium phosphate buffer, pH 7, with 25 μM Ru(bpy)₃ ²⁺ and were recorded at 25 mV/s. Figure 8 illustrates the additive nature of the electrochemical response from the guanine oxidation and the carbon nanotube oxidation. The d(T)₆o wrapping sequence contains no oxidizable guanines and there is no current enhancement in the cyclic voltammograms for the samples which contain the metal mediator and oligonucleotide only with respect to the cyclic voltammograms of the metal mediator alone (not shown). Current above the mediator only background is observed for the oligonucleotides, d(T)₅o(G)i_O, and d(GT)₃₀, in the absence of CNT due to the electrochemical oxidation of the guanine bases by the soluble metal mediator Ru(bpy)₃ ²⁺. The inclusion of the guanine base into the wrapping sequence results in an increase in signal as the number of guanines in the wrapping sequence is increased. The concentration of the oligonucleotide strand in the suspended CNT solution was very similar for all of the guanine containing wrapping sequences (Table 4), but as the guanine content increased, the guanine concentration in solution also increased leading to the increased electrochemical response observed for both the d(T)_5O(G)io and d(GT)₃o oligonucleotide only samples and the d(T)_5O(G)io and d(GT)₃₀- wrapped CNTs as shown in Figures 7 and 8. When the currents observed for the oligonucleotide only solutions and the solutions of the DNA-wrapped CNT (Figure 7) are compared, it is obvious that the signal from the both guanine oxidation and from the CNT oxidation are observed and the signals are additive, which has important implications for inclusion of CNT in biosensing applications.

Example 8 Analysis of Chonoamperometric Response of DNA-Wrapped CNT:

Inclusion of Guanine in the Wrapping Sequence Chronoamperometry. The chronoamperometric responses were recorded for Ru(bpy)₃ ²⁺ mediated oxidation of (1(T)₆₀, d(T)₅₉(G)i, d(T)₅₀(G)₁₀ and d(GT)₃₀- wrapped CNT at three concentrations of CNT (0.005, 0.01 and 0.02 mg/mL). For each CNT concentration there is a corresponding oligonucleotide concentration in the as-prepared solutions (Table 4). The plots of the CA response versus t^1/2 for the four different wrapping sequences at three concentrations of carbon nanotubes all show a biphasic response. For each curve there are two linear regimes: an early, fast regime and a slower regime at longer times. The two regimes were interpreted as two phases of the electrocatalytic reaction and the apparent second order rate constants were determined from the slope of the best fit linear lines at the early and late times with the slope of the line equal to (πk'C_z ^*)^1/2.¹² Similar behavior has been seen in numerous cases with oxidation of guanine in DNA.¹¹ The rate constants obtained in the presence and absence of CNT are reported in Table 5, in the columns labeled +CNT and -CNT, respectively. The rate constants were calculated for all CNT concentrations, but only the results for the CNT concentration of 0.02 mg/mL are given in Table 5. As expected, the rate constants found were independent of CNT and guanine concentration.

As shown by the cyclic voltammetry results, the electrochemical response obtained in the presence of CNTs is a combination of the oxidation of the CNT and the guanine base (if present) in the wrapping sequence, so the response reflects the combined rate of oxidation for both species. The concentration used to calculate the rate constants from the slope of the best-fit line was a combination of the CNT concentration and the guanine concentration measured in each solution. The CNT concentrations were calculated using a concentration equivalent to the active site concentration of the CNT from the estimate of 1 electron for every 5 nm length of carbon nanotube calculated by Diner,et al. from redox titration experiments, assuming an average length of 140 nm for each carbon nanotube.^1"3 The guanine concentration is calculated based on the measured concentration of oligonucleotide strand in solution times the number of guanines in the specific wrapping sequence. The guanine concentration used in the calculation of the rate constants, given in Table 5, does not distinguish between guanines that are wrapped on CNTs or free in solution. The rate constants obtained in the absence of CNT are for samples containing oligonucleotide only.

Table 5: CA Generated Second-Order Rate Constants for the Oxidation of dd((TT))₆₆₀₀,, dd((TT))_5s99((GG))ii,, dd((TT))_550O((GG))₁,₀o aanndd dd(GT)₃₀-wrapped CNTs by Ru(bpy)₃ ²⁺ for a 0.02 mg/mL Concentration of CNT

n/a - Not applicable because no oxidizable guanines are present in the d(T)₆₀ sequence.

The biphasic nature of the CA traces strongly supports the idea that the metal- mediated oxidation for the DNA-wrapped CNT is a complex, multi-electron process. (Note that the rate constants for the CNT are expressed in terms of 5 nm reducing equivalents, not the entire carbon nanotube.) The apparent second-order rate constants obtained for both the fast and slow phases of the biphasic CA curve increase as the number of guanines in the wrapping sequence is increased. This observation is most apparent at the 0.02 mg/mL CNT concentration (Table 5), where the rate constants for the fast phase vary from 5 x 10⁶ M^'V for the d(T)₆₀-wrapped CNT to 16 x 10⁶ M^'V¹ for the d(GT)3o- wrapped CNT, a threefold increase. The fast rate constants obtained support the theory of fast electron transfer from both the guanine base and the carbon nanotube to Ru(bpy)₃ ³⁺. One would expect that if the concentrations of both of the oxidizable species are adequately described by the model, the rate constants calculated should be independent of sequence; however, a three- fold increase in rate constants calculated from the fast portion of the CA trace is observed for the electrocatalytic reaction of d(GT)₃₀ sequence compared to the d(T)₆₀ sequence. This result suggests an increased reactivity of the guanine base in the wrapping sequence as the number of guanines in the sequence is increased, which could result from enhanced electronic coupling between the guanine base and the carbon nanotube. For the slow phase of the reaction, the difference in the rate constants is only apparent for the d(GT)₃₀ wrapped CNT at the 0.02 mg/mL concentration. All of the other wrapping sequences give similar rate constant values for the slow phase in the presence of carbon nanotube. In addition, the rate constant calculated from the CA response for the guanine oxidation in the absence of CNT (~4 x 10⁶ M^-1S^"1) is similar to that seen in previous work.¹¹'¹⁴

Example 9

Analysis of Cyclic Voltammograms of DNA-Wrapped CNT:

Inclusion of the Guanine in the Wrapping Sequence

Digital simulation of cyclic voltammograms. The rate constants derived from the CA responses given in Table 5 were also obtained by simulations of the CV data collected for the same carbon nanotube and guanine concentrations. The experimental data and the simulations for the Ru(bpy)₃ ²⁺-mediated oxidation of the d(T)₆₀, d(T)₅o(G)io and d(GT)₃₀-wrapped CNT are shown in Figure 9 for CNT concentrations of 0.005, 0.01 and 0.02 mg/mL. The homogeneous electron transfer rate constants (k_f) derived from the simulated data at CNT concentrations of 0.005, 0.01 and 0.02 mg/mL and the respective guanine concentration (Table 4) are found in

Table 6.

To simulate the electrocatalytic signal from the metal-mediated oxidation of guanine and CNT, an EC mechanism was used. The following form was employed to simulate the carbon nanotube oxidation:

Ru(bpy)₃ ²⁺ >- Ru(bpy)₃ ³⁺ + e- (E)

Ru(bpy)j³⁴ + CNT >^■ Ru(bpy)₃ ²⁺ + CNT_0x (C)

The following form was employed to simulate the guanine oxidation: Ru(bpy)₃ Ru(bpy)₃ ³⁺ + e^' (E)

2+

Ru(bpy)₃ ³⁺ + G Ru(bpy)₃~ + G_o: (C)

Table 6: Simulated Rate Constants for d(T)₆₀, d(T)₅₀(G)i₀ and d(GT)₃₀-wrapped CNTs at 0.005, 0.01 and 0.02 mg/mL

Multiple C reaction steps were needed to adequately fit the electrocatalytic signal generated from the oxidation reaction of the carbon nanotubes to account for additional signal from the multiple electron transfers from a DNA-wrapped CNT. This approach has been used in the simulation of DNA electrocatalysis when a single homogeneous electron transfer rate constant (from 1 C reaction) cannot account for the observed signal.¹¹'^13"16 Addition of C reactions to the mechanism helped to account for the continued reactivity of a CNT after the initial chemical electron transfer step. As observed previously,¹ ten C reactions were used to approximate the electrochemical response of metal-mediated CNT oxidation. Two C reactions were used to approximate the electrochemical response of the metal-mediated guanine oxidation, as we have done previously.¹¹'¹³'¹⁶ The number of successive C reactions and the value of their homogeneous electron transfer rate constants were determined by optimizing the shape and intensity of the simulation to produce a good fit for the voltammogram. The decrease in k_f values with successive C reaction in the mechanism produced a good simulation of the experimental voltammogram.

The oxidation of the guanine base by soluble metal mediators has been studied in the past. As observed here, the initial electron transfer is relatively facile, and the extraction of a second electron is possible but occurs at a rate much slower than the first electron transfer.¹¹'¹³'¹⁶ The rate constant calculated from the simulations for the guanine oxidation in the absence of CNT in these studies is very fast, 1-2 x 10⁶ M^-1S^"1 for d(T)_5O(G)io and 2-6 x 10⁶ M^'V¹ for d(GT)₃₀ and similar to that observed for the fast portion of the CA trace. The increasing value of the rate constant with increasing guanine content may reflect an increase in the number of productive collisions that result in electron transfer.¹⁶ The first rate constants, Ic₁, determined by simulating the data for the three wrapping sequences at a 0.02 mg/mL CNT concentration range from 7 x 10⁶ for the d(T)₆₀-wrapped CNT to 2O x IO⁶ M^'V¹ for the d(GT)₃₀- wrapped CNT, very similar to the rate constants derived from the fast portion of the CA trace and also exhibiting the three-fold increase in rate with increased guanine concentration (Table 6). The rate constants that describe the later reaction steps are very similar to the rate constants derived from the slower portion of the CA trace and show a slight increase in the rate constants for the d(GT)₃o-wrapped CNT at 0.02 mg/mL. (For the d(T)₆₀-wrapped CNT, the nine rate constants for the later reactions range form 3 x 10³ to 6 x 10⁶ M^"'s^{" 1} and those for the d(GT)₃₀- wrapped CNT range from 8 x 10⁴ to 1 x 10⁷ M^-1S^"1). This analysis provides some insight into the electron transfer kinetics for Ru(bpy)₃ ²⁺- mediated CNT oxidation: the approach used for simulating CNT oxidation suggests that the initial electron transfer is fast while slower, successive electron transfers are important for evaluating the observed electrocatalytic signal. The lower values of the homogeneous electron transfer rate constants for later C reactions in the simulation mechanism suggest that there is a limit to which a given CNT can be successively oxidized. The availability of these additional electrons from CNT likely arises from the formation of oxidized functionalities on the surface of the nanotube. An increase in the C rate constants is observed as the number of guanines in the wrapping sequence increases. This is most prominent at the 0.02 mg/mL CNT concentration. As observed in the rate constants calculated from the fast portion of the CA trace, the rate constants for the first reaction step, ki, range from 6.5 x 10⁶ M^{" 1}S^"1 for the d(T)₆₀-wrapped CNT to 2O x IO⁶ M ¹S^"1 for the d(GT)₃₀-wrapped CNT, a three-fold increase. As suggested above, the increasing value of the rate constant with increasing guanine content may reflect the increase in productive collisions that result in electron transfer¹⁶ or the possibility of enhanced electronic coupling between the CNT and guanine.

Example 10

Impact of Ionic Strength on Electrocatalytic Detection of Carbon Nanotubes

To assess the impact of ionic strength the electrochemical responses for a d(T)₆o-wrapped CNT and d(GT)₃₀- wrapped CNT were recorded in solutions of 0.1 M sodium phosphate buffer, pH 7 with and without added NaCl. The ionic strength of the buffer solution strongly influences the strength of the metal mediator binding to the DNA. At the higher ionic strengths, the binding of the metal mediator is significantly weaker than at the lower ionic strengths due to the shielding of the negative charge on the backbone of the nucleic acid by the Na⁺. The weaker binding in the higher ionic strength solution reduces the overall catalytic signal when compared to the lower ionic strength solution, because guanine oxidation occurs primarily through the bound Ru(bpy)₃ ³⁺.

Shown in Figure 1OA are the cyclic voltammograms of 25 μM Ru(bpy)₃ ²⁺, d(T)₆₀ and d(T)₆₀- wrapped CNT at 0.01 mg/mL CNT in the lower ionic strength buffer consisting of 0.1 M sodium phosphate, pH 7 and in the higher ionic strength buffer consisting of 0.1 M sodium phosphate, pH 7 plus 0.8 M NaCl. Because the d(T)₆o oligonucleotide sequence contains no oxidizable guanines there is little to no current enhancement for the samples which contain the metal mediator and the oligonucleotide only with respect to cyclic voltammograms of the metal mediator in either buffer. Also, because the d(T)₆₀ contains no oxidizable guanines there is no difference in the signal between the low and high salt buffers. The overall catalytic signal of the d(T)₆o-wrapped CNT was also relatively unaffected by the ionic strength of the buffer, suggesting that the current observed was a result of direct contact between Ru(bpy)₃ ³⁺ and the carbon nanotube and did not require strong binding to the wrapped DNA for abstraction of an electron. The current enhancement observed for the DNA-wrapped CNT is due to oxidation of the CNT by electrogenerated Ru(III) and is independent of the ionic strength of the buffer solution. Similar results were observed for the d(T)₃o and d(T)i₂₀- wrapped CNT.

Shown in Figure 1OB are the cyclic voltammograms of 25 μM Ru(bpy)₃ ²⁺, d(GT)₃₀ and d(GT)₃₀-wrapped CNT at 0.01 mg/mL CNT in the lower ionic strength buffer consisting of 0.1 M sodium phosphate, pH 7 and in the higher ionic strength buffer consisting of 0.1 M sodium phosphate, pH 7 plus 0.8 M NaCl. Because the d(GT)₃o oligonucleotide sequence contains thirty oxidizable guanines there is electrocatalytic signal above the Ru(bpy)₃ ²⁺ background for the oligonucleotide only samples. A decrease in current for the samples which contain the metal mediator and the oligonucleotide in high salt buffer is observed with respect to cyclic voltammograms of the metal mediator and the oligonucleotide in low salt buffer. Again, the decrease in the catalytic signal in the high ionic strength buffer is due to the weaker binding of the metal mediator to the DNA backbone. The overall catalytic signal of the d(GT)₆₀-wrapped CNT did decrease, but that decrease can be attributed to the reduction of signal from guanine oxidation. The signal due to the CNT oxidation appeared to be unaffected by the ionic strength of the buffer, suggesting that the current observed was a result of direct contact between Ru(bpy)₃ ³⁺ and the carbon nanotube and did not require strong binding with the wrapped DNA for abstraction of an electron. The current enhancement observed for the DNA- wrapped CNT is due to oxidation of the CNT by electrogenerated Ru(III) and is independent of the ionic strength of the buffer solution.

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The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. A method of detecting a carbon nanotube, comprising:

(a) reacting said carbon nanotube with a first transition metal complex that oxidizes said carbon nanotube in a first oxidation-reduction reaction, regenerating the reduced form of said first transition metal complex in a catalytic cycle; and

(b) detecting said carbon nanotube by detecting said first oxidation-reduction reaction.

2. The method of claim 1, wherein said carbon nanotube is dispersed in a solution.

3. The method of claim 2, wherein said solution is an aqueous solution.

4. The method of claim 1 , wherein said carbon nanotube is complexed with a nucleic acid.

5. The method of claim 4, wherein said nucleic acid is selected from the group consisting of single stranded DNA, double stranded DNA, RNA and PNA.

6. The method of claim 1, wherein said carbon nanotube is coupled to a solid support.

7. The method of claim 6, wherein said solid support comprises an electrode and a nonconductive layer, and said carbon nanotube is coupled to said nonconductive layer.

8. The method of claim 1, wherein said carbon nanotube is selected from the group consisting of multi -walled nanotubes and single-walled nanotubes.

9. The method of claim 1, wherein, wherein said transition metal complex is selected from the group consisting of Ru(bpy)₃ ²⁺, Ru(Me₂ -bpy)₃ ²⁺, Ru(Me₂ -phen)₃ ²⁺, Fe(bpy)₃ ²⁺, Fe(5-Cl-ρhen)₃ ²⁺, Os(5-Cl-phen)₃ ²⁺, and ReO₂ (py)₄ ¹⁺-

10. The method of claim 1, wherein said detecting step is carried out by cyclic voltammetry, normal pulse voltammetry, chronoamperometry, or square-wave voltammetry.

11. A method of detecting a target compound, comprising:

(a) coupling said target compound to a carbon nanotube;

(b) reacting said carbon nanotube with a first transition metal complex that oxidizes said carbon nanotube in a first oxidation-reduction reaction, regenerating the reduced form of said first transition metal complex in a catalytic cycle; and (c) detecting said target compound by detecting said first oxidation-reduction reaction.

12. The method of claim 11, wherein said target compound is a first member of a binding pair, wherein said carbon nanotube further comprises a second member of a specific binding pair, and wherein said coupling step is carried out by specifically binding first and second members of said binding pair.

13. The method of claim 11, wherein said target compound is covalently coupled to said carbon nanotube.

14. The method of claim 11, wherein said target compound is a protein, peptide or nucleic acid.

15. The method of claim 11, wherein said carbon nanotube is dispersed in a solution.

16. The method of claim 11, wherein said carbon nanotube is complexed with a nucleic acid.

17. The method of claim 11, wherein said carbon nanotube is coupled to a solid support.

18. The method of claim 11, wherein said carbon nanotube is selected from the group consisting of multi-walled nanotubes and single-walled nanotubes.

19. The method of claim 11, wherein, wherein said transition metal complex is selected from the group consisting of Ru(bpy)₃ ²⁺, Ru(Me₂ -bpy)₃ ²⁺, Ru(Me₂ -phen)₃ ²⁺,

FeOw)₃ ²⁺, Fe(5-Cl-phen)₃ ²⁺, Os(5-Cl-phen)₃ ²⁺, and ReO₂ (ρy)₄ ¹⁺.

20. The method of claim 11, wherein said detecting step is carried out by cyclic voltammetry, normal pulse voltammetry, chronoamperometry, or square-wave voltammetry.