An inhibition type alkaline phosphatase biosensor for amperometric determination of caffeine
Introduction
Caffeine is a naturally occurring substance found in the leaves, seeds or fruits of some plant species and is a member of a group of compounds known as methylxanthines. It is also present in many painkillers and antimigraine pharmaceuticals [1], [2]. The most commonly known sources of caffeine are coffee, cocoa beans, cola nuts and tea leaves. It does not accumulate in the body over the course of time and is normally excreted within several hours of consumption.
It shows various physiological effects on different body systems, including the central nervous, cardiovascular, gastrointestinal, respiratory and renal systems [3]. It is a powerful stimulant of the central nervous system and also stimulates the cardiac muscle. However, high amounts of this alkoloid cause a noticable irritation of the gastrointestinal tract as well as other undesired effects. Higher doses of caffeine can cause anxiety, jitteriness, and upset stomach [4]. Caffeine can exaggerate the body's response to stress by increasing heart rate and blood pressure [5]. Caffeine increases calcium excretion in the urine and so heavy caffeine usage may increase the risk of osteoporosis [6]. The levels of caffeine in different matrices such as biological, pharmaceutical, and herbal have been determined by numerous techniques including UV spectrophotometric [7], [8], derivative spectrophotometric [9], voltammetric [10], [11], piezoelectric [12], [13], HPLC [14], [15], [16], [17], [18], [19], [20], capillary electrophoresis (CE) [21], FTIR [22], NIR reflectance spectrometry [23], caffeine sensitive membrane electrode [24], MS [25], and also GC/MS methods [26], [27], pH electrode [28], whole cells based biosensor [29], and a voltammetric sensor based on molecularly imprinted polymer [30].
Spectrophotometry is a fast and simple method. It is not possible to determine caffeine directly in coffee beans by conventional UV absorption measurement due to the spectral overlap. On the other hand, the derivative spectrophotometer is relatively easy; however, it is not reliable for the determination of small concentrations of caffeine in samples. HPLC methods use expensive equipments and they need operator attention for their applications in the laboratories. Other methods such as FT infrared and NIR reflectance spectrometry are equally versatile for the measurement of caffeine and do not require expensive chemicals, however, such instruments are expensive and are not available in most laboratories.
According to the literature data, caffeine shows an inhibition effect on the activity of alkaline phosphatase [31], [32], [33]. By considering these data we have developed an inhibition type alkaline phosphatase biosensor for determination of caffeine. From the literatures there is no any study based on the inhibition of alkaline phosphatase to determine caffeine, so, the purpose of this work is to develop a novel screen printed biosensor based on the inhibition of alkaline phosphatase for direct and simple determination of caffeine in beverages without using pre-separation or background correction procedures.
Section snippets
Chemicals
Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa, glutaraldehyde (25%), cysteamine, p-nitrophenyl phosphate disodium salt hexahydrate, caffeine, potassium chloride, magnesium chloride, KH2PO4, K2HPO4, glycine and all the other chemicals were purchased from Sigma Chemical Co. (USA).
Electrochemical experiments were carried out in glycine buffer (50 mM; pH 10.5 containing 0.1 M KCl + 1 mM MgCl2). Enzyme solution was prepared in Tris–HCl buffer (pH 7.0).
Apparatus
Electrochemical measurements were
Determination of the inhibition effect of caffeine on ALP
For determination of the inhibition effect of caffeine on ALP some cyclic voltammetric experiments were carried out. CV measurements were performed between + 0.3 and +1.1 V in glycine buffer (pH 10.5, 50 mM). Measurements were carried out for bare electrode, ALP modified electrode in the presence of p-nitrophenyl phosphate without caffeine, and ALP modified electrode in the presence of p-nitrophenyl phosphate with caffeine. Voltammograms obtained from the experiments were given in Fig. 1. From
Conclusion
In this study, an amperometric biosensor based on alkaline phosphatase was developed in order to investigate the effect of caffeine on the activity of alkaline phosphatase enzyme. From the experimental studies we detected an inhibition effect of caffeine on the enzyme activity and this effect increased at higher caffeine concentrations. Using the biosensor we detected a linear concentration range for caffeine in the presence of a constant concentration of p-nitrophenylphosphate. The developed
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