Nitrogen monoxide (NO) is an unstable radical species of a short life, and has been elucidated to have important functions as a physiological active substance in a living body (Chemistry Today [Gendai Kagaku], April, 1994, Special Edition; Pharmacia, May, 1997, Special Edition). Methods for measuring nitric oxide are roughly classified into indirect methods, which measure NO.sub.2.sup.- and NO.sub.3.sup.- as oxidative degradation products of nitric oxide, and methods based on direct measurement of nitric oxide. The direct methods have been eagered from viewpoints of detection and quantification of nitric oxide under physiological conditions. However, any specific and highly sensitive detection method that can be applied to in vitro systems has not been developed so far.
As typical methods, there have been known, for example, the chemiluminescence method utilizing the luminescence generated by ozone oxidation of NO radicals (Palmer R. M., et al., Nature, 327, pp.524-526, 1987), a method determining absorption spectrum of metHb which is produced by oxidation of oxyhemoglobin (O.sub.2 Hb) (Kelm M., et al., Circ. Res. 66, pp.1561-1575, 1990), a method for quantification utilizing the flow of electric current produced in oxidation when electrodes are placed in a tissue (Shibuki K., Neurosci. Res. 9, pp.69-76, 1990; Malinski, and T., Nature, 356, pp.676-678, 1992), the Griess reaction method (Green L. C., et al., Anal. Biochem., 126, pp.131-138, 1992) and so forth (as reviews, see, "Approaches From The Newest Medicine [Saishin Igaku Kara No Approach] 12, NO", Ed. by Noboru Toda, pp.42-52, Section 3, Tetsuo Nagano, Measuring Method of NO, published by Medical View Co., Ltd; Archer, S., FASEB J., 7, pp.349-360, 1993).
The Griess reaction method achieves the detection by using azo coupling of a diazonium salt compound and naphthylethylenediamine in the presence of NO.sub.2.sup.- that is produced by oxidation of a nitric oxide radical. Although this method does not achieve direct measurement of nitric oxide radicals, the method has the merit of requiring no special apparatuses or techniques. Moreover, this method also has a characteristic feature that nitric oxide-related metabolites can be quantified, since NO.sub.3.sup.- can be measured by reduction to NO.sub.2.sup.- with cadmium (Stainton M. P., Anal. Chem., 46, p.1616, 1974; Green L. C., et al., Anal. Biochem., 126, pp.131-138, 1982) or hydrazine (Sawicki, C. R. and Scaringelli, F. P., Microchem. J., 16, pp.657-672, 1971).
As a reagent for measuring nitric oxide by detecting NO.sub.2.sup.- in a similar manner to the Griess reaction method, 2,3-diaminonaphthalene has been known. This reagent reacts with NO.sub.2.sup.- under acidic conditions to form a fluorescent adduct, naphthalenetriazole (chemical name: 1-[H]-naphtho[2,3-d]triazole, Wiersma J. H., Anal. Lett., 3, pp.123-132, 1970). The conditions for the reaction of 2,3-diaminonaphthalene with NO.sub.2.sup.- have been studied in detail, demonstrating that the reaction proceeds most quickly at pH 2 or lower and completes in approximately 5 minutes at room temperature (Wiersma J. H., Anal. Lett., 3, pp. 123-132, 1970; Sawicki, C. R., Anal. Lett., 4, pp.761-775, 1971). Furthermore, the generated adduct emits fluorescence most efficiently at pH 10 or higher (Damiani, P. and Burini, G., Talanta, 8, pp.649-652, 1986).
The measurement of nitric oxide using the 2,3-diaminonaphthalene is characterized in that a detection limit is about several tens nanomoles and sensitivity is 50 to 100 times higher than that of the Griess reaction method (Misko, T. P., Anal. Biochem. 214, pp.11-16, 1993). Moreover, the method is also excellent in that it can be carried out conveniently without requiring any special apparatuses or techniques (as a review of the above description, see, DOJIN News, No. 74, Information Measurement Reagents for NO: 2,3-Diaminonaphthalene, published by Dojindo Laboratories Co., Ltd., 1995). However, since this method does not utilizes nitric oxide, per se, but its oxidation product, NO.sub.2.sup.-, as the reaction species, the method is rather indirect as compared to the direct method for measuring nitric oxide. In addition, since the reaction of 2,3-diaminonaphthalene and NO.sub.2.sup.- is performed under strongly acidic conditions (pH 2 or lower), it has a problem that the method cannot be available for detection and quantification of nitric oxide under a physiological condition.
The inventors of the present invention conducted researches to provide means for direct measurement of nitric oxide with high sensitivity under a physiological condition. As a result, the inventors found that 2,3-diaminonaphthalene or derivatives thereof efficiently reacts with nitric oxide to give fluorescent naphthalenetriazole or its derivatives, even under a neutral condition, in the presence of an oxygen source such as dissolved oxygen or oxide compounds (for example, PTIO and its derivatives such as carboxy-PTIO). Moreover, the inventors also found that a method for measuring nitric oxide employing this reaction gave extremely high detection sensitivity and achieved accurate quantification of a trace amount of nitric oxide (see, the specification of Japanese Patent Application No. 7 189978/1995).
However, the aforementioned method utilizing 2,3-diaminonaphthalene needs irradiation by excitation light of a short wavelength such as about 370 to 390 nm for the detection of fluorescence, and accordingly, cells and tissues in a measurement system may possibly be damaged. The method also has a problem in that strong autofluorescence of cells may affect the measurement and, in the fluorescence measurement, excitation light cannot be sufficiently cut with a fluorescence filter equipped on usual fluorescence microscopes. Moreover, the fluorescent triazole compound produced from 2,3-diaminonaphthalene does not have sufficient fluorescence intensity and, for this reason, it is difficult to accurately measure fluorescence in individual cells by conventional fluorescence microscopes. In addition, there is also a problem that 2,3-diaminonaphthalene itself is not suitable as a basic structure for various chemical modification so that the reagent can be localized inside of cells because of its simple chemical structure.
It has recently been reported that certain fluorescein derivatives, which themselves do not emit substantial fluorescence, can readily react with nitric oxide under a neutral condition to form a triazole compound exhibiting fluorescence of high intensity, and the triazole derivative can emit strong fluorescence of about 515 nm by means of long wavelength excitation light of approximately 495 nm (Kojima et al., the 16th Medicinal Chemistry Symposium, the 5th Annual Meeting of the Pharmaceutical Chemistry Section, the Lecture Abstracts, pp.166-167, Subject No. 2-P-26, published by the Pharmaceutical Society of Japan, Oct. 23, 1996).
When these fluorescein derivatives are used as an agent for measurement of nitric oxide, excitation light can be easily cut by a fluorescence filter provided on usual fluorescence microscopes, and intracellular nitric oxide concentration can easily be measured by observing fluorescence in individual cells. However, the fluorescence wavelength range of the aforementioned fluorescein derivatives partly overlaps with the autofluorescence wavelength range of cells and, accordingly, it may sometimes be impossible to accurately quantify nitric oxide. Moreover, since the fluorescence may be attenuated under acidic conditions (e.g., pH 4 or lower), there is also a problem that accurate measurement cannot be performed in a wide pH range.
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