Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-05
2001-07-17
Sisson, Bradley L. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S183000, C435S189000, C435S287200, C204S450000, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06261780
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for quantitatively analyzing a biochemical compound utilizing electrochemcial reaction.
BACKGROUND OF THE INVENTION
Biochemical compounds such as glucose, cholesterol, urea nitrogen, uric acid, bilirubin, ammonia, hemoglobin, neutral fat, lactic acid, fructosylamino acid, and L-glycerolphosphoric acid are contained in various biological liquids or body fluids such as whole blood, plasma, serum, urine, lymph, and pulpal liquid. The quantitative analysis of the biochemical compound (i.e., analyte) in body fluids is of great value for diagnosis of patients suffering from various diseases.
Previously, the quantitative analysis of the analytes have been performed by a wet method or a dry method. The wet method generally comprises a color formation reaction between the analyte and a color-forming reagent or in the presence of the analyte in an aqueous medium. The dry method generally employs a strip or a multilayer analytical element containing a color-forming reagent. Each of these methods has each advantageous feature and therefore both are widely employed in diagnosis of patients who possibly suffer from various diseases. However, both methods have disadvantageous features in that it is not easy to obtain the test results quickly because the color formation reaction is involved, and further they show a relatively low sensitivity in analyzing an extremely small amount of analyte.
In order to obtain the test results more quickly and to analyze an extremely small amount of analyte with an increased sensitivity, and enzyme sensor such as glucose sensor has been developed. The analytical system of the glucose sensor comprises the step of oxidizing the target glucose with glucose oxidase to produce gluconic acid and hydrogen peroxide and the step of detecting the amount of the produced hydrogen peroxide by a hydrogen peroxide electrode to give an electric signal corresponding to the amount of the hydrogen peroxide. This system is disadvantageous from the view point of complicated apparatuses involved.
In Chemical Letters pp. 989-990 (1998), a report entitled “Enhanced Electron Transfer from Glucose Oxidase to DNA-Immobilized Electrode aided by Ferrocenyl Naphthalene Diimide, a Threading Intercalator” is published. The author includes the present inventors. The report discloses that a ferrocene-modified 1,4,5,8-naphthalene-tetracarboxydiimide bound to double stranded DNA by a threading intercalation mode, enhanced the electron transfer between glucose oxidase and a DNA-immobilized electrode. This report, however, does not teach that the discovery may be utilized in analytical methods.
Recently, it has been desired to provide a method of quantitative analysis of analytes in body fluids which gives the analytical results more easily and more quickly using a simple apparatus.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the invention to provide a method for quantitative analyzing analytes in body fluids which gives the analytical results quickly and easily using a simple apparatus.
The present invention resides in a method for quantitatively analyzing a biochemical compound which comprises bringing the biochemical compound into contact with double stranded DNA fragments which are fixed onto a surface of an electrode at their one terminals and in which electrochemically active threading intercalators are intercalated, in an aqueous medium under application of electric potential to the electrode in the presence of an oxidase capable of oxidizing the biochemical compound and becoming a reduced oxidase, and detecting an electric current flowing between the electrode and another electrode placed in the aqueous medium.
In the analytical method of the invention, examples of the biochemical compounds include glucose, cholesterol, lactic acid, fructosylamino acid and L-glycerolphosphoric acid, and examples of the oxidases include glucose oxidase, cholesterol oxidase, lactose oxidase, fructosylamino acid oxidase and L-glycerolphosphoric acid oxidase.
In the analytical method, the double stranded DNA fragments can be formed by combining, by hybridization, complementary DNA fragments with single stranded DNA fragments fixed onto the surface of electrode at their one terminals or by attaching separately prepared double stranded DNA fragments to the surface of electrode.
In the analytical method, the electrochemically active threading intercalator preferably is a compound showing oxidation-reduction activity, and more preferably is a compound having two chain groups at each terminal of which a ferrocene moiety is attached. The electrochemically active threading intercalator is present preferably in the aqueous medium in a concentration of 10 nM to 10 mM. The double stranded DNA fragments are fixed onto the electrode surface preferably in an amount of 10
−11
to 10
−10
mol. per 1 mm
2
of the electrode surface.
Ogawa Masashi
Takagi Makoto
Takenaka Shigeori
Fuji Photo Film Co. , Ltd.
Reed Smith LLP
Sisson Bradley L.
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