Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2000-02-18
2002-10-22
Whisenant, Ethan C. (Department: 1655)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S006120, C435S091100, C435S287100, C536S023100, C536S024300
Reexamination Certificate
active
06468785
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention (Technical Field)
The present invention relates to methods, compositions and devices for the detection of nucleic acids utilizing electrochemical detection with polymer-coated electrodes.
2. Background Art
The biological significance of nucleic acids has required the development of novel analytical methods for their detection and quantitation. Historical methods involve use of gel electrophoresis with radioactive labeled probes. Other label methods have been used, including biotin, digoxigenin and fluorescent dyes. These methods all involve long detection times and complex laboratory procedures, with ancillary amplification methods, such as polymerase chain reaction (PCR), frequently required to produce sufficient materials for detection.
In some applications, microscale separation techniques such as capillary electrophoresis are coupled to optical detection techniques for identification of individual nucleic acids. UV absorbance and laser-induced fluorescence systems have been used for this purpose. Electrochemical detectors, while used for other bioanalytical applications (Wang, J. Analytical Electrochemistry, VCH Publishers, New York, 1994; U.S. Pat. No. 5,516,644), have received only limited attention for nucleic acid analysis. However, electrochemical detector analysis systems, if demonstrated to be accurate and reproducible, offer potential advantages for detection of DNA and RNA and for “lab-on-a-chip” devices, including high sensitivity and selectivity, ultra-small dead volumes, fast response, compatibility with advanced microfabrication and miniaturization technologies, low-cost, and minimal power requirements.
Electrochemical detection of DNA has traditionally relied on the electroactivity of nucleobases. In particular, the oxidation of the purine bases at carbon electrodes has been exploited for amperometric detection of nucleic acids in flow-injection (Wang, J.; Chen, L.; Chicharro, M.
Anal. Chim. Acta,
319, (1996) 347) and capillary-electrophoresis (Xu, D. K.; Hua, L.; Chen, H. Y.
Anal. Chim. Acta,
335, (1996) 95) systems. Another electrochemical route under evaluation relies on the oxidation of the sugar backbone at copper surfaces (Singhal, P.; Kuhr, W. G.
Anal. Chem.,
69, (1997) 4828). Such detection schemes can be used for monitoring both purine- and pyrimidine-containing nucleic acids, but require the use of an alkaline medium and specialized sinusoidal voltammetric instrumentation. In addition to these direct anodic detection schemes, several groups have explored indirect electrochemical protocols for detecting DNA (Takenaka, S.; Uto, Y.; Kondo, H.; Ihara, T.; Takagi, M.
Anal. Biochem.,
218, (1994) 436; Woolley, A. T.; Lao, K.; Glazer, A.; Mathies, R. A.
Anal. Chem.,
70, (1998) 684).
One application for biosensing devices is the in situ detection of DNA hybridization. Methods and devices for combining the base-pair recognition of DNA probes with the advantages of electrochemical transducers are currently receiving significant attention due to numerous potential applications (E. K. Wilson,
Chem. Eng. News, May
25, 1998, 47; S. R. Mikkelsen,
Electroanalysis
8(1996) 15). Most of these devices rely on measuring changes in the peak current of a redox-active marker that preferentially binds to the duplex formed in the hybridization event (K. M. Millan, S. R. Mikkelsen,
Anal. Chem.
65(1993) 2317; K. Hashimoto, K. Ito, Y. Ishimori,
Anal. Chem.
66(1994) 3830). Label-free electrochemical detection of hybridization reactions represents a very attractive approach for detecting DNA sequences (H. Korri-Youssoufi, F. Garnier, P. Srivastava, P. Godullot, A. Yassar,
J. Am. Chem. Soc.
119(1997) 7388; P. Bauerle, A. Emge,
Adv. Mater.,
10(1998) 324; J. Wang, G. Rivas, J. Fernandes, L. Paz, M. Jiang, R. Waymire,
Anal. Chim. Acta
375(1998) 197; and E. Souteyrand, J. P. Cloarec, J. R. Martin, C. Wilson, I. Lawrence, S. Mikkelsen, M. F. Lawrence,
J. Phys. Chem. B
101(1997) 2980). Such approaches rely on monitoring changes in electronic or interfacial properties accompanying the DNA hybridization event. Label-free detection thus greatly simplifies the sensing protocol, since it eliminates the need for indicator addition, association and detection steps, and potentially offers instantaneous detection of the duplex formation.
Conducting-polymer molecular interfaces hold particular promise for inducing electrical signals resulting from DNA interactions. Changes in the properties of conducting polymers accompanying DNA hybridization have been reported in connection with the electropolymerization of oligonucleotide-substituted films (Korri-Youssoufi et al., supra; Bauerle et al., supra; U.S. Pat. No. 5,837,859). However, all such reported methods have been indirect, such as the method of Korri Youssoufi et al., which employs functionalized conjugated polymers, with an amino-substituted oligonucleotide grafted on a precursor copolymer, or the method of U.S. Pat. No. 5,837,859, employing copolymerization utilizing a covalent bond, such as with a spacer arm. Other methods, such as that of U.S. Pat. No. 5,156,810, employ a polymerized surfactant layer incorporating a ligand on a substrate. Other patents of interest include U.S. Pat. No. 5,776,672, which employs secondary substrates and requires approximately one hour per assay. The immobilization of DNA onto conductive surfaces is of enormous interest both in studies of DNA itself and numerous applications ranging from DNA diagnostics to gene therapy. A key requirement for such investigations and applications is the achievement of an efficient interface between the nucleic acid system and the conductive surface.
SUMMARY OF THE INVENTION (DISCLOSURE OF THE INVENTION)
In one embodiment, the invention is an apparatus for detection of DNA hybridization, which apparatus includes a specimen electrode with a detection surface and a conducting polymer composition coating at least a portion of the detection surface of the specimen electrode, wherein the conducting polymer composition comprises a conducting polymer and a free oligomer complementary to the DNA sequence to be detected. The oligomer is free in that the oligomer is not conjugated or bonded to the conducting polymer or any secondary substrate. The specimen electrode may be a carbon, metallic or metal-coated crystal electrode. The conducting polymer may be an electropolymerized substance, such as polypyrrole, polythiophene, polyaniline or a derivative thereof. The free oligomer can be an oligonucleotide from about 8 to about 50 mers, and preferably from about 20 to about 30 mers.
The apparatus for detection of DNA hybridization can include means for accumulating a specimen on at least a portion of the polymer coated detection surface of the specimen electrode and means for amperometric detection of the specimen electrode upon accumulation of a specimen on at least a portion of the polymer coated detection surface of the specimen electrode. Means for accumulating a specimen can include a reservoir or other receptacle or volume, and can also include pumps, shunts, tubes and other structure for accumulating a specimen. Means for amperometric detection can include any metering or measuring device or system for amperometric and other electric current-related measurements, which may be digital or analog, and which may be optionally integrated into a computer-based system.
The apparatus for detection of DNA hybridization can further include a reference electrode and means for determining the change in potential of the specimen electrode relative to the reference electrode upon accumulation of a specimen on at least a portion of the polymer coated detection surface of the specimen electrode.
In another embodiment, the invention provides a method for detection of DNA in a test specimen, including the steps of:
providing a specimen electrode with a detection surface;
coating at least a portion of the detection surface with a conducting polymer composition, which composition includes a conducting polymer and a free oligom
Fortes Antonio
Jiang Mian
Mukherjee Baidehi
Wang Joseph
Lu Frank
New Mexico State University Technology Transfer Corporation
Ownbey Nancy E.
Slusher Stephen A.
Whisenant Ethan C.
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