Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-24
2002-02-12
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091500, C435S091510, C536S023100, C600S345000
Reexamination Certificate
active
06346387
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the detection of biological substances through binding interactions and, in particular, to methods of detecting proteins and other substances by mediated catalytic electrochemistry.
2. Description of the Related Art
For many reasons, researchers are interested in the detection of biological substances such as nucleic acids, proteins, and carbohydrates. Detection of such biomolecules can allow for identification and development of targets for drug discovery and gene expression analysis. The electrochemical detection of nucleic acids provides an alternative to fluorescent and radiochemical detection techniques that potentially eliminates the need for labeling.
The parent applications of the instant application, whose entire specifications, drawings, and claims are specifically incorporated herein by reference, disclose, among other inventions, sequencing and methods of qualitatively and quantitatively detecting nucleic acid hybridization. Such inventions represent a major advance in the art and provide oxidation-reduction reactions that function in a catalytic manner without the addition of an enzyme or fluorescent label. These catalytic reactions are useful for determining the presence or absence of nucleic acids and provide for extremely accurate testing of biological samples. More specifically, catalytic oxidation has been found to be useful for quantitative detection of preselected nucleic acid bases (U.S. Pat. No. 5,871,918). The disclosures of each of the patents and publications referred to herein are incorporated herein by reference.
The technology described in U.S. Pat. No. 5,871,918 utilizes the discovery that nucleotide bases of DNA can be electrochemically oxidized using transition metal complexes as mediators. In this system, the nucleotide bases function as an array of endogenous redox-active labels that allow for ultrasensitive detection of DNA in conjunction with microelectrode methods. The detection reaction follows a two-step mechanism involving reversible oxidation/reduction of the mediator. First the mediator is oxidized by an electrode. Then, the mediator is reduced by the preselected nucleotide base and reoxidized at the electrode. In order for mediated oxidation of nucleic acids to proceed efficiently, the mediator and nucleotide base should have similar oxidation potentials. For example, catalytic oxidation of guanine can be carried out using the mediator, ruthenium
2+
(2,2′-bipyridine)
3
(Ru(bpy)
3
2+
). In solution, Ru(bpy)
3
2+
exhibits a reversible redox couple at 1.05 V (vs. Ag/AgCl reference), similar to the oxidation potential observed for guanine (about 1.1 V vs. Ag/AgCl). Thus, addition of guanine-containing DNA to a solution of Ru(bpy)
3
2+
leads to catalytic enhancement in the electrochemical oxidation current via the following reaction sequence:
Ru(bpy)
3
2+
→Ru(bpy)
3
3+
+e-
Ru(bpy)
3
3+
+DNA →DNA
ox
+Ru(bpy)
3
2+
where DNA
ox
represents a DNA molecule in which guanine has undergone a one electron oxidation.
The regeneration of reduced Ru(bpy)
3
2+
by reaction with guanine creates a catalytic cycle in which the presence of DNA is detected by transfer of electrons from the preselected base to the electrode. The number of turnovers obtained in the catalytic cycle depends on the number of electrons in the preselected base that can be oxidized by the mediator and the number of preselected bases. In the case of guanine oxidation, Ru(bpy)
3
2+
is capable of oxidizing guanine by at least two electrons (Armistead, P. M. and Thorp, H. H.,
Anal. Chem
. 2000, 72, 3764), and some reports suggest as many as 30 electrons obtained from guanine through overoxidation steps (Thorp, H. H.,
Trends Biotechnol
. 1998, 16, 117). A typical DNA molecule will contain on average about one guanine every four bases so even a small oligonucleotide will have multiple guanines available for catalytic turnover of Ru(bpy)
3
2+
. As a result of these properties, detection of nucleic acids via mediated catalytic electrochemistry is an extremely sensitive method.
Thus, in one embodiment of the prior invention, a nucleic acid sample is contacted with an oligonucleotide probe, which possesses a sequence, at least a portion of which is capable of binding to a known portion of the sequence in the nucleic acid sample, to form a hybridized nucleic acid, after which the hybridized nucleic acid is reacted with a suitable mediator, which is capable of oxidizing a preselected nucleic acid base in the hybridized nucleic acid sample in an oxidation-reduction reaction.
The selection of mediator in this prior work is dependent upon the particular preselected nucleotide base chosen, and is readily determinable by those skilled in the art. Particularly preferred mediators include transition metal complexes that are capable of participating in electron transfer with the preselected base such that the reduced form of the metal complex is regenerated, completing a catalytic cycle. An example of a suitable transition metal complex is Ru(bpy)
3
2+
; however, the mediator or oxidizing agent may be any molecule such as a cationic, anionic, non-ionic, or zwitterionic molecule that is reactive with the preselected base at a unique oxidation potential to transfer electrons from the nucleic acid to the electrode. All that is required is that the mediator be reacted with the hybridized nucleic acid sample under conditions sufficient to achieve the selective oxidation of the preselected base.
The oxidation-reduction rate is detected, for example, with a detection electrode, and the electronic signal may be detected by cyclic voltammetry or other means known in the art. Hybridized DNA target contains guanine and is therefore more redox-active than the probe strand, which preferably is either selected or designed to contain a minimal number of guanines.
In U.S. Pat. No. 5,968,745 of Thorp et al., a polymer-electrode is provided that is useful for the electrochemical detection of a preselected base in a nucleic acid. The polymer-electrode comprises: (a) a substrate having a conductive working surface; and (b) a polymer layer on the conductive working surface. The polymer layer has a plurality of microfluidic reaction openings distributed throughout the layer. An oligonucleotide probe is preferably bound to the polymer layer.
U.S. Pat. No. 6,127,127 provides a self-assembled phosphonate monolayer, which in the preferred embodiment is a carboxy-alkyl phosphonate, on an ITO surface. The oligonucleotide probe is immobilized on an electrode surface modified by the self-assembled monolayer. The electrode with the self-assembled monolayer is useful for the electrochemical detection of a preselected base in a nucleic acid and for determining the presence of a target nucleic acid in a sample, by contacting the self-assembled monolayer with the sample, so that the target nucleic acid and the oligonucleotide probe form a hybridized nucleic acid on the monolayer; reacting the hybridized nucleic acid with a transition metal complex capable of oxidizing a preselected base in the hybridized nucleic acid in an oxidation-reduction reaction; detecting the oxidation-reduction reaction; and determining the presence or absence of the target nucleic acid from the detected oxidation-reduction reaction.
In both the polymer-electrode and monolayer patents, determination of the presence of a target protein in a sample can also be achieved and comprises attaching a protein-binding substance to a polymer-electrode or self-assembled monolayer on a conductive working surface according to the invention; exposing the polymer-electrode or monolayer to the sample; exposing the polymer-electrode or monolayer to a second protein-binding substance that has been modified to contain an oligonucleotide label; reacting the polymer-electrode or monolayer with a transition metal complex capable of oxidizing a preselected base in the oligonucleotide label in an oxidat
Eckhardt Allen E.
Groelke John W.
Stewart David H.
Thorp H. Holden
Chakrabarti Arun Kr.
Fredman Jeffrey
Myers Bigel & Sibley & Sajovec
Xanthon, Inc.
LandOfFree
Detection of binding reactions using labels detected by... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Detection of binding reactions using labels detected by..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Detection of binding reactions using labels detected by... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2975264