Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1998-12-23
2001-08-21
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S164000, C436S512000, C436S805000, C436S829000, C436S905000, C435S007100, C435S287100, C435S287200, C435S287900, C435S808000, C435S969000, C530S300000, C424S143100
Reexamination Certificate
active
06277652
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a technique for analyzing different ligands (analytes) in biosamples, and more specifically relates to a novel colorimetric sensor comprising a polydiacetylene membrane and an analysis method which employs the sensor.
BACKGROUND OF THE INVENTION
Membranes formed by self-assembling of amphipathic (amphiphilic) diacetylene molecules exhibit a blue color when polymerized with UV (ultraviolet) irradiation, and such polydiacetylene membranes are known to undergo a change in color to red by the effects of pH, temperature increase, mechanical stress, etc. (see for example, Lipowsky, R. (1991) Nature 349, 475-481; Bloor, D. and Chance, R. R. (1985) Polydiacetylenes: NATO ASI Series E, Applied Science).
Recently, applications of polydiacetylene membranes as biosensors utilizing this property have been proposed (Charych, D. H. et al., (1993) Science 261, 585-588; Reichert, A. et al., (1995) J. Am. Chem. Soc. 115, 1146-1147 (1995); Charych D. H. et al., (1996) Chemistry & Biology 3, 113-120 (1996)). In particular, attempts have been made to construct biosensors in such a manner that the receptors which react specifically with pathogenic bacteria, viruses, toxins and the like present in biosamples are incorporated into polydiacetylene membranes, and the color change (blue to red) induced when the receptors bind to their specific ligands (pathogenic bacteria, viruses, toxins, etc.) is utilized to allow detection of the ligands with high sensitivity. To date, only saccharides and lipids have been used as the receptors in such proposed methods.
Such methods, however, can only be applied to the detection of ligands wherein the binding structure of the receptor and ligand is known and the receptor has been identified. Therefore a number of receptors must be synthesized which is equivalent to the number of types of ligands to be detected, presenting the likely insurmountable difficulty that the conditions for preparation of the polydiacetylene membrane must be determined for the respective cases. Many receptors consisting of saccharides and lipids are highly complicated and difficult to synthesize, while the color change for detection of the ligand is often insufficient. In the method of Charych, et. al. (Chemistry & Biology 3, 113-119 (1996)), for example, gangliosides are incorporated as the receptors for detection of the influenza virus. Because of insufficient color change of the polydiacetylene membrane upon binding between the polydiacetylene membrane and the influenza virus, however, it is necessary to introduce sialic acid into the polydiacetylene at a few percent as a structural change promoter. This complicates the process for preparation of the membrane as a sensor. When the ligands to be detected are different, other types of substances for promoting the structural change must be designed.
In addition, the conventional methods are generally only effective for ligands for which the receptors have molecular weight of about 1000 or less, and the methods are not suitable for detecting ligands which bind to macromolecular receptors. This is because the macromolecular receptors cause color changes in polydiacetylene membranes by simply being incorporated therein.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a new analysis system utilizing polydiacetylene membranes for analyzing a variety of biosamples in a simple yet highly sensitive manner, wherein the preparation of the membranes is facilitated.
The present inventors were led to the present invention by the finding that the abovementioned object can be attained by incorporating into a polydiacetylene membrane a relatively low molecular weight protein capable of reaction or interaction with a ligand (analyte) in a biosample.
Thus, the present invention provides a colorimetric sensor characterized by comprising polydiacetylene membrane liposomes, a polydiacetylene membrane film or fine particles coated with a polydiacetylene membrane, in which said polydiacetylene membrane is incorporated with a protein having a reduced molecular weight low enough not to cause color change in the polydiacetylene membrane.
In a preferred embodiment, the reduced-molecular-weight protein in the colorimetric sensor of the invention is an antibody Fab′ fragment which undergoes an antigen-antibody reaction with an antigen contained in a sample. In another preferred embodiment, the reduced-molecular-weight protein in the colorimetric sensor of the invention is an antigenic protein of molecular weight of 100,000 or less which undergoes an antigen-antibody reaction with an antibody contained in a sample. In still another preferred embodiment, the reduced-molecular-weight protein in the colorimetric sensor of the invention is a peptide consisting of 3-20 amino acid residues which undergoes an antigen-antibody reaction with an antibody contained in a sample. In still another preferred embodiment, the reduced-molecular-weight protein in the colorimetric sensor of the invention is a combination of a single-stranded DNA of 100 bases or less which hybridizes with single-stranded DNA contained in a sample to form a double-stranded DNA, and an antibody which reacts with the double-stranded DNA but does not react with the single-stranded DNA contained in the sample.
The invention also provides a method for analysis of a biosample, which comprises contacting the abovementioned colorimetric sensor with a solution sample and utilizing an absorption measurement or a visual observation with the naked eye to detect color change in the polydiacetylene membrane.
The sensor of the present invention which comprises a polydiacetylene membrane is suitable for wide use and allows highly sensitive and simple detection of various ligands including ligands (analytes) which cannot be detected by the prior art. The colorimetric sensor of the invention can be manufactured easily under similar conditions for different target ligands.
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“A litmus test' for molecular recognition using artificial membranes,” by Charych et al., Chemistry & Biology 1996, vol. 3, No. 2.
“Polymerized Liposomes Containing C-Glycosides of Sialic Acid: Potent Inhibitors of Influenza Virus in Vitro Infectivity” J. Am. Chem. Soc. 1993, 115, 1146-47.
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Inoue Toshiki
Jo Yoshio
Takada Kouichi
Chin Christopher L.
Do Pensee T.
Fay Sharpe Fagan Minnich & McKee LLP
Hogy Medical Co., Ltd.
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