Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-30
2001-10-16
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091200, C436S094000, C536S023100
Reexamination Certificate
active
06303316
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the field of detection and identification of analytes, using novel compositions and apparatus comprising one or more nucleic acid ligands operably coupled to an organic semiconductor. More particularly the present invention relates to methods of use of organic semiconductor chip technology, flow cell technology or magnetic filter separation technology, along with compositions comprising nucleic acid ligands, to identify analytes. The present invention further relates to the detection, identification and neutralization of chemical and biological warfare agents.
2. Description of Related Art
There is a great need for the development of methods and apparatus capable of detecting and identifying unknown chemical and biological agents (herein referred to as analytes), which include but are not limited to nucleic acids, proteins, illicit drugs, explosives, toxins, pharmaceuticals, carcinogens, poisons, allergens and infectious agents. Possible approaches to this problem include the use of nucleic acid microchip technology or magnetic bead technology. Although these technologies are known for various applications (e.g., Hacia et al, 1996; Shoemaker et al., 1996; U.S. Pat. Nos. 3,970,518; 4,230,685; 4,677,055; 4,695,393; 5,861,242; 5,578,832), the present invention provides a novel and unexpected use of these technologies to detect and identify unknown analytes.
As one skilled in the art will readily appreciate, any method, technique or device capable of such detection and identification would have numerous medical, industrial and military applications. For instance, such methods, techniques and devices could be employed in the diagnosis and treatment of disease, to develop new compounds for pharmaceutical, medical or industrial purposes, or to identify chemical and biological warfare agents.
Current methods, techniques and devices that have been applied to identification of chemical and biological analytes typically involve capturing the analyte through the use of a non-specific solid surface or through capture deoxyribonucleic acids (DNA) or antibodies. A number of known binding agents must then be applied, particularly in the case of biological analytes, until a binding agent with a high degree of affinity for the analyte is identified. A labeled antiligand (e.g., labeled DNA or labeled antibodies) must be applied, where the antiligand causes, for example, the color or fluorescence of the analyte to change if the binding agent exhibits affinity for the analyte (i.e., the binding agent binds with the analyte). The analyte may be identified by studying which of the various binding agents exhibited the greatest degree of affinity for the analyte.
There are a number of problems associated with current methods of chemical and biological agent identification. It takes a great deal of time and effort to repetitiously apply each of the known labeled antiligands, until an antiligand exhibiting a high degree of affinity is found. Accordingly, these techniques are not conducive to easy automation. Current methods are also not sufficiently robust to work in the heat, dust, humidity or other environmental conditions that might be encountered, for example, on a battlefield or in a food processing plant. Portability and ease of use are also problems seen with current methods for chemical and biological agent identification.
SUMMARY OF THE INVENTION
The present invention fulfills an unresolved need in the art, by providing a recognition complex and a recognition complex system that are capable of identifying an unknown chemical or biological agent (hereafter, “analyte”) and, if desired, identifying and amplifying a neutralizing agent capable of inactivating or destroying the analyte. The recognition complex and recognition complex system and the corresponding techniques should be capable of full automation.
In one embodiment, the recognition complex system of the present invention employs organic semiconductor chip technology, wherein nucleic acid ligands are distributed across the surface of the chip so as to form an array of recognition complexes, each recognition complex comprised of a nucleic acid ligand attached to an organic semiconductor. In a preferred embodiment, the organic semiconductor is DALM (diazoluminomelanin), although the use of other organic semiconductors, such as polyphenylenes, is contemplated within the scope of the invention. For certain applications, the recognition complexes may be positioned between a pair of electrodes. Binding of analyte to a recognition complex may be detected by changes in the electrical or photochemical properties of the nucleic acid ligand/organic semiconductor couplet upon binding to the analyte. The degree to which the electrical and photochemical properties change is a function of the degree to which the nucleic acid ligand binds the analyte. Accordingly, the electrical and photochemical changes that occur across all of the recognition complexes, when taken as a whole, can be used as a unique signature to identify the analyte.
In certain embodiments, the analyte to be identified may be added to the array in the form of a complex mixture that may include, for example, aqueous or organic solvent, proteins, lipids, nucleic acids, detergents, particulates, intact cells, bacteria, viruses and spores, as well as other components. In other embodiments, the analyte may be partially or fully purified before exposure to the array.
In another embodiment, the nucleic acid ligand sequences that bind to the analyte may be isolated, amplified (e.g., using a polymerase chain reaction) and redistributed across a clean chip surface and attached to the organic semiconductor to form a new array. The nucleic acid ligand sequences that do not bind to the analyte may be discarded. The new array is exposed to the analyte and binding of analyte to nucleic acid ligands produces an enhanced electrical and photochemical signature, as the nucleic acid ligand sequences present on the new array more specifically compliment the analyte. This procedure may be repeated, with each iteration producing a more unique or enhanced signature.
In a further embodiment, this iterative process may be used to identify and amplify one or more nucleic acid ligand sequences that exhibit the highest degree of affinity for the analyte. Production of a nucleic acid ligand that binds to the analyte with high affinity (dissociation constant of 1.0 &mgr;M or lower) would have utility in a variety of applications. For certain embodiments, production of a nucleic acid ligand with a dissociation constant of 10 nM or lower is preferred.
In another embodiment, nucleic acid ligands that bind to the analyte with high affinity can be reproduced (synthesized or amplified) for use as a neutralizing agent to inactivate or destroy the analyte. A high affinity nucleic acid ligand may be attached to a variety of agents that could be used to neutralize the analyte. In certain embodiments, the high affinity nucleic acid ligand can be attached to an organic semiconductor, such as DALM. The DALM
ucleic acid ligand couplet, after binding to the analyte, may be activated by a variety of techniques, including exposure to sunlight, heat, or irradiation of various types, including laser, microwave, radiofrequency, ultraviolet and infrared. Activation of the DALM
ucleic acid ligand couplet results in absorption of energy, which may be transmitted to the analyte, inactivating or destroying it. It is contemplated within the scope of the invention that the nucleic acid ligand could be attached to other agents that would inactivate the analyte, such as toxic proteins, enzymes capable of activating protoxins, or other molecules or reactive moieties including organic or inorganic compounds.
In other embodiments, the high affinity nucleic acid ligand could be incorporated into an apparatus capable of being carried into the field, for example, by soldiers or vehicles entering a battlefield. As an example, the high affinity nucleic acid ligand
Alls John L.
Batishko Charles R.
Bruno John G.
Holwitt Eric A.
Kiel Johnathan L.
Blakely & Sokoloff, Taylor & Zafman
Conceptual Mind Works, Inc.
Horlick Kenneth R.
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