Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
2000-06-15
2003-08-05
Nguyen, Nam (Department: 1753)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C205S792000, C204S403010, C204S412000
Reexamination Certificate
active
06602400
ABSTRACT:
BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION
This invention relates to the detection of biomolecules. Specifically, the invention relates to an apparatus and methods for efficient, high-throughput electrical or electrochemical detection of biomolecules. In addition, the invention relates to such apparatuses having manipulation electrodes associated with test sites for enhancing concentration or reaction of a biomolecule at a test site, with or without providing detection electrodes at the test sites. In embodiments having detection electrodes, the manipulation electrodes increase the occurrence of a desired bio-conjugation event at a test site, while the detection electrodes detect the occurrence of the desired bio-conjugation event. Methods for using the apparatuses of the invention, particularly for analysis of nucleic acids, are also provided.
2. Background of the Invention
A number of commonly-utilized biological applications, including for example, diagnoses of genetic disease, analyses of sequence polymorphisms, and studies of receptorligand interactions, rely on the ability of analytical technologies to readily detect events related to the interaction between biomolecules. These detection technologies have traditionally utilized fluorescent compounds or radioactive isotopes to monitor such interactions. For example, Potyrailo et al., 1998
, Anal. Chem
. 70: 3419-25, describe an apparatus and method for detecting interactions between immobilized fluorescently-labeled aptamers and peptides. There are, however, significant disadvantages associated with the use of radioactive or fluorescent labels to track interactions between biomolecules, including heightened health risks and increased experimental cost and complexity.
Methods for electrical or electrochemical detection of molecular interactions between biomolecules have provided an attractive alternative to detection techniques relying on radioactive or fluorescent labels. Electrical and electrochemical detection techniques are based on the detection of alterations in the electrical properties of an electrode arising from interactions between one group of molecules attached to the surface of an electrode (often referred to as “probe” molecules) and another set of molecules present in a reaction mixture (often referred to as “target” molecules) contacted with the electrode. Methods and devices related to electrical or electrochemical detection of biomolecules are disclosed in U.S. Pat. Nos. 4,072,576, 4,098,645, 4,414,323, 4,840,893, 5,164,319, 5,187,096, 5,194,133, 5,312,527, 5,532,128, 5,591,578, 5,653,939, 5,670,322, 5,705,348, 5,770,369, 5,780,234, 5,824,473, 5,891,630, 6,017,696 and International Application, Pub. No. WO 97/01646.
Electrical or electrochemical detection eliminates many of the disadvantages inherent in use of radioactive or fluorescent labels to detect interactions between the probe and target molecules. This process offers, for example, a detection technique that is safe, inexpensive, and sensitive, and is not burdened with complex and onerous regulatory requirements.
The development of microfabricated arrays of biomolecules (microarrays) has led to further improvements on traditional analytical techniques for the detection of molecular interactions between biomolecules. Microarrays of biomolecules (e.g., oligonucleotides, nucleic acids, proteins, peptides, or antibodies) have utility in a wide variety of applications in which molecular interactions between target molecules in a reaction mixture and large numbers of distinct probe molecules bound to defined regions of a substrate can be simultaneously assayed using electrical, optical, or radioactive detection strategies. Microarrays, therefore, satisfy the demand for inexpensive, high-throughput detection of biomolecular interactions.
Although microarrays have proven to be useful for high-throughput detection of molecular interactions between biomolecules, microarrays have proven to be inefficient with respect to reaction time. The probability that a particular target molecule will bind to an immobilized probe molecule is determined by the concentration of the target molecule in a reaction mixture, the diffusion rate of the target molecule, and the binding affinity of the target molecule for the immobilized probe molecule. Since target molecules in many diagnostic assays are only present in minute quantities (e.g., <10
−12
M), interactions between target molecules and immobilized probe molecules therefore occur infrequently, and reaction times of several days are not uncommon.
In recognition of the inherent inefficiency of microarrays for detecting interactions between biomolecules, several techniques for increasing the rate of molecular interactions between biomolecules (termed “bio-conjugation events” herein) have been proposed. These techniques typically involve attaching a probe molecule to an electrode, which is then electrically biased to attract the target molecule through enhancing ion migration transport. For example, U.S. Pat. No. 5,653,939 to Hollis et al. discloses a method for detecting hybridization between a target molecule in a sample solution and an oligonucleotide probe bound at a test site, wherein an electric potential is applied to an electrode comprising the test site. However, current flow through the electrode causes electrolysis at the test site, which will reduce rather than enhance bio-conjugation events at such sites.
U.S. Pat. Nos. 5,605,662; 5,632,957; and 6,017,696 to Heller et al. disclose methods for controlling molecular biological reactions in microscopic formats that utilize a self-addressable, self-assembling microelectronic apparatus. Heller et al. further provide an apparatus in which target molecules labeled with fluorescent dyes are transported by free field electrophoresis to specific test sites where the target molecules are concentrated thereby, and reacted with specific probes bound to that test site. Unbound or non-specifically interacting target molecules are thereafter removed by reversing the electric polarity at the test site. Interactions between probe and target molecules are subsequently assayed using optical means. Heller et al., however, do not suggest using their devices for electrical or electrochemical detection of molecular interactions.
There remains a need in the art to develop more efficient, high-throughput devices and methods for the detection of molecular interactions between biomolecules. In particular, there remains a need in the art to develop more efficient, high-throughput devices and methods for electrical or electrochemical detection of molecular interactions. More particularly, there remains a need in the art to develop electrical or electrochemical detection devices in which the occurrence of a desired bio-conjugation event at a test site can be favorably manipulated, thereby yielding a device with increased throughput. The development of such devices, and methods for their use, would have wide application in the medical, genetic, and molecular biological arts.
SUMMARY OF THE INVENTION
The present invention provides an apparatus and methods for efficient, high-throughput detection of bio-conjugation events occurring at a test site. The invention specifically provides apparatuses comprising a pair of manipulation electrodes at a test site to enhance occurrence of said bio-conjugation events. In particular embodiments, the apparatuses of the invention also comprise detection electrodes at a test site for electrical or electrochemical detection of bio-conjugation events occurring at the test site. In the apparatuses of the invention, the detection and manipulation electrodes are separate and distinct, and are arranged in the apparatus to avoid electrolysis at the test site. In one embodiment of the apparatuses of the invention, the arrangement of the manipulation electrodes is such that electrolysis is prevented from occurring at the manipulation electrodes. In another particular embodiment of the invention, the arrangement of the manipulation electrodes in th
Choong Vi-En
Li Changming
Maracas George
Shi Song
Dorsey & Whitney LLP
Motorola Inc.
Nguyen Nam
Olsen Kaj K.
Silva Robin M.
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