Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
2001-01-12
2002-10-22
Tung, T. (Department: 1743)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C204S403140, C204S403100, C204S403060
Reexamination Certificate
active
06468416
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a biosensor by which the concentration of L-phenylalanine contained in various samples can be quickly and conveniently quantitated without resort to any troublesome pretreatments. More particularly speaking, it relates to a biosensor which is useful in quantitating L-phenylalanine contained in biological samples (blood, urine, saliva, sweat, etc.), food samples and the like by an electrochemical measurement method with the use of L-phenylalanine dehydrogenase, etc. This method is particularly significant in newborn mass screening for detecting phenylketonuria (PKU), which is an amino acid metabolic error, at the early stage, or monitoring the daily living of patients suffering form this disease.
BACKGROUND OF THE INVENTION
L-Phenylalanine is an important amino acid which is one of the essential amino acids and contained in a large amount of biological samples as well as in foods and drinks employed as L-phenylalanine sources. On the other hand, the disease known as PKU is one of typical hereditary amino acid metabolic errors wherein tyrosine is not synthesized due to L-phenylalanine dehydrogenase deficiency and thus L-phenylalanine is pooled at an abnormally high level in the blood. When allowed to stand, PKU induces serious intellectual disturbance, speech disturbance, amelanotic symptom, etc.
To prevent this disease, newborn mass screening has been widely carried out in Japan and foreign countries. As a typical example of methods for determining L-phenylalanine concentration in the blood, the Guthrie method with the use of dried blood spot has been employed and has largely contributed to early diagnoses.
Patients with PKU thus found should have a diet therapy restricting their L-phenylalanine intake. Namely, such patients should have specially prepared dishes with the elimination or reduction of L-phenylalanine at least until reaching the majority, preferably throughout their lives. Since L-phenylalanine is one of the essential amino acids for the human-body, the L-phenylalanine concentration should be strictly regulated so that it can be taken at the maximum level without inducing brain disturbance, etc. but yet at the minimum level required for the growth of the body. In treating PKU, it is therefore essential to monitor not only the phenylalanine level in the blood but also the phenylalanine intake in the daily diet.
Under these circumstances, examples of methods for determining L-phenylalanine by using blood as samples include the liquid chromatographic method (Journal of Chromatography, Vol. 274, p. 318 (1983)) and the bioassay method with the use of dried blood spot, i.e., the method widely known as the Guthrie method (Pediatrics, Vol. 32, p. 338 (1963)). In the former method it is necessary to subject samples to a specific pretreatment and to use expensive measuring instruments. Although the latter method can be conveniently carried out, it takes a long time to complete the reaction and the results need to be scrutinized with the naked eye. Namely, each of these methods suffers from some problems from the viewpoint of convenience or rapidness in quantification.
There have been also reported determination methods with the use of enzymes, for example, a method with the use of L-phenylalanine ammonia-lyase (Methods of Enzymatic Analysis, Vol. 8, p. 405 (1985)), a method with the use of L-phenylalanine oxidase (Clinica Chimica Acta, Vol. 136, p. 131 (1984)) and a method with the use of L-phenylalanine dehydrogenase (Japanese Laid-Open Patent Publication No. 63-129996(A)). In these documents, the usefulness of these methods in quantification is pointed out. In particular, the method of determining phenylalanine in the blood with the use of L-,phenylalanine dehydrogenase is reported in detail by Hummel et al. (Analytical Biochemistry, Vol. 170, p. 397 (1988)) and Wendel et al. (Analytical Biochemistry, Vol. 180, p. 91 (1989), Clinica Chimica Acta, Vol. 192, p. 165 (1990)). In each of these methods, the blood employed as a,sample is subjected to various pretreatments and then an enzyme reaction is carried out. In addition, the quantification cannot be performed unless expensive and relatively large-scale measuring instruments (a spectrophotometer, etc.) are employed.
In recent years, biosensors, in particular, sensors with the use of enzymes have been vigorously developed. Through them all, it has become possible to conveniently and highly accurately determine blood glucose level as disclosed in EP 351891 and WO 86/07632. Although attempts have been recently made by Huang et al. to determine L-phenylalanine (Analytical Chemistry, Vol. 70, p. 991 (1998)), there still remain a number of problems to be solved in putting the sensor to practical use.
In this method, a carbon paste containing L-phenylalanine dehydrogenase mixed with two other enzymes (i.e., salicylate hydroxylase and tyrosinase) is filled in a tube (1.2 mm×1.5 mm×30 mm) which is provided with a copper wire as a lead wire. After polishing the electrode surface, it is employed as a working electrode. A detection unit composed of this working electrode, a reference electrode made of silver/silver chloride (Ag/AgCl) and a counter electrode made of platinum is connected to a reactor to thereby fabricate a determination unit. The determination is carried out by mixing a buffer, a salicylic acid solution and oxidized nicotinamide adenine dinucleotide (NAD
+
) in the reactor, adding a sample, and calculating the L-phenylalanine concentration by using a computer from the response current obtained after the reaction. Thus phenylalanine in a concentration range of from 20 to 150 &mgr;M can be quantitated and the detection sensitivity is 5 &mgr;M. However, there still remain problems to be solved in, for example, the stability of the response and the storage stability of the enzyme electrode thus constructed.
Although the biosensor technology as described above is just making it possible to determine L-phenylalanine by using L-phenylalanine dehydrogenase, a quick and convenient quantification is still impossible since it is necessary in this method to prepare reagents and instruments and perform troublesome procedures and operations. Although it is desirable for patients with PKU that L-phenylalanine can be conveniently determined at home so as to monitor the blood L-phenylalanine level and examine foods and drinks to be cooked, it is still difficult at the present stage.
Under these circumstances, it is an object of the present invention to provide a biosensor, by which L-phenylalanine can be highly accurately, quickly and conveniently quantitated without resort to the use of many reagents, large-scaled measuring devices or instruments, troublesome pretreatments or special techniques, and a determination method therefor.
SUMMARY OF THE INVENTION
To achieve the above-described object, the present invention provides an L-phenylalanine sensor composed of an electrode system, which comprises at least a working electrode and a counter electrode formed on an insulating support, and a reagent reaction layer, which contains as reaction reagents at least L-phenylalanine dehydrogenase, NAD
+
or oxidized nicotinamide adenine dinucleotide phosphate (NADP
+
) as a coenzyme and an electron mediator and is integrated with the above electrode system, and a method of determining L-phenylalanine by which L-phenylalanine. can be electrochemically quantitated simply by adding a sample to the above-described sensor without resort to any troublesome pretreatments.
In the L-phenylalanine sensor according to the present invention, an absorbent carrier containing as reaction reagents at least L-phenylalanine dehydrogenase, NAD
+
or NADP
+
and an electron mediator is located as a reagent reaction layer between the electrodes of an electrode system, which comprises at least a working electrode and a counter electrode provided opposite to each other on an insulating support, and integrated with the electrode system. Owing to this structure, both of the enzyme reac
Nakamura Kenji
Shinozuka Naoki
Yokoyama Toru
Noguerola Alexander
Sapporo Immuno Diagnostic Laboratory
Tung T.
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