Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
1998-08-14
2001-09-18
Tung, T. (Department: 1743)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C204S403060, C204S450000, C204S452000, C204S409000, C204S600000, C204S603000
Reexamination Certificate
active
06290839
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to compositions and methods useful in the electrophoretic transport of target analytes to a detection electrode comprising a self-assembled monolayer (SAM). Detection proceeds through the use of an electron transfer moiety (ETM) that is associated with the target analyte, either directly or indirectly, to allow electronic detection of the ETM.
BACKGROUND OF THE INVENTION
There are a number of assays and sensors for the detection of the presence and/or concentration of specific substances in fluids and gases. Many of these rely on specific ligand/antiligand reactions as the mechanism of detection. That is, pairs of substances (i.e. the binding pairs or ligand/antiligands) are known to bind to each other, while binding little or not at all to other substances. This has been the focus of a number of techniques that utilize these binding pairs for the detection of the complexes. These generally are done by labelling one component of the complex in some way, so as to make the entire complex detectable, using, for example, radioisotopes, fluorescent and other optically active molecules, enzymes, etc.
Other assays rely on electronic signals for detection. Of particular interest are biosensors. At least two types of biosensors are known; enzyme-based or metabolic biosensors and binding or bioaffinity sensors. See for example U.S. Pat. Nos. 4,713,347; 5,192,507; 4,920,047; 3,873,267; and references disclosed therein. While some of these known sensors use alternating current (AC) techniques, these techniques are generally limited to the detection of differences in bulk (or dielectric) impedance.
The use of electrophoresis in microfluidic methods to facilitate the binding of biological molecules to their binding partners for subsequent detection is known; see for example U.S. Patent Nos. 5,605,662 and 5,632,957, and references disclosed therein.
Similarly, electronic detection of nucleic acids using electrodes is also known; see for example PCT US 97/20014 and U.S. Pat. No. 5,770,369.
However, there is a need for a system that combines electrophoretic transport of target analytes, including nucleic acids, to a detection electrode for subsequent electronic detection.
SUMMARY OF THE INVENTION
In accordance with the objects outlined above, the present invention provides methods of detecting a target analyte in a sample comprising placing the sample in an electric field between at least a first electrode and at least a second electrode sufficient to cause electrophoretic transport of the sample to a detection surface. The detection surface comprises a detection electrode comprising a monolayer comprising conductive oligomers, and optionally a capture binding ligand capable of binding the target analyte. The target analyte directly or indirectly binds to the capture binding ligand to form a binding complex. The binding complex further comprises at least one ETM. The presence of the ETM is detected using the detection electrode. Optionally, the first electrode can comprise a permeation layer.
In an additional aspect, the invention provides methods comprising placing the sample in an electric field between at least a first electrode and at least a second electrode sufficient to cause electrophoretic transport of the sample to a detection surface. The detection electrode comprises a monolayer comprising conductive oligomers, and a capture probe. The target sequence directly or indirectly hybridizes to the capture probe to form a first hybridization complex, wherein the hybridization complex further comprises at least one ETM. The presence of the ETM is detected using the detection electrode.
In an additional aspect, the invention provides methods of detecting a target nucleic acid sequence in a sample comprising placing the sample in an electric field between at least a first electrode and at least a second electrode sufficient to cause electrophoretic transport of the sample to a detection surface comprising a detection electrode comprising a monolayer comprising conductive oligomers and a capture probe. The method further comprises directly or indirectly attaching at least one label probe to the target sequence to form an assay complex comprising at least one ETM. The presence of the ETM is detected using the detection electrode.
In an additional aspect, the invention provides electronic devices adapted to receive a sample solution comprising charged target analytes. The device comprises a first electrode comprising a permeation layer, a second electrode, and a detection electrode. The detection electrode comprises a monolayer comprising conductive oligomers, and a capture binding ligand capable of binding the target analyte. The first and second electrodes are in electrical contact upon the introduction of the sample solution such that electrophoretic transport of the charged target analytes to the detection electrode will occur.
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