An inhibition type alkaline phosphatase biosensor for amperometric determination of caffeine

doi:10.1016/j.electacta.2010.04.038

Electrochimica Acta

Volume 55, Issue 18, 15 July 2010, Pages 5195-5199

https://doi.org/10.1016/j.electacta.2010.04.038 Get rights and content

Abstract

A biosensor based on the inhibition of alkaline phosphatase (ALP) enzyme was developed for caffeine determination. The biosensor was prepared by a chemical covalent immobilization of alkaline phosphatase with a cross-linking agent, glutaraldehyde on a ceramic based gold screen printed electrode that was modified with cysteamine by forming a self-assembled monolayer. Caffeine competitively inhibits ALP enzyme and the determination method of caffeine by the biosensor was based on this inhibition effect of caffeine. The principle of the measurement was based on the determination of the differentiation of biosensor responses in the enzymatic reaction catalyzed by ALP in the absence and the presence of caffeine. Differences between the biosensor responses were related to caffeine concentration which was added in to the reaction medium. Caffeine concentration can be determined accurately between 0.1 and 10 μM using the biosensor. Detection limit of the biosensor is 0.08 μM. In the optimization studies of the biosensor, glycine buffer (pH 10.5; 50 mM) and 30 °C were obtained as the optimum working conditions.

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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The standard method for ALP activity determination in clinical fluids is based on a spectrophotometric assay, in which the sample is mixed with an alkaline buffer and a substrate (para-nitrophenyl phosphate), and measuring the product at 405 nm (Keiding et al., 1974). This substrate is also widely used in electrochemical biosensors (Akyilmaz and Turemis, 2010). Recently, our group reported an electrochemical biosensor for the determination of ALP in clinical samples (Sappia et al., 2019).
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This article presents an electrochemical impedance study on the detection of Alkaline Phosphatase (ALP) in phosphate buffer saline (PBS) solution by using anti-ALP functionalized Si nanowires (SiNWs). Electrochemical impedance was measured using a three-electrode cell where the working electrode was a very dense and highly disordered array of Si NWs, e.g. SiNW-forest. This array was grown using plasma enhanced chemical vapor deposition (PECVD) at 350 °C. The NW surface was modified by immobilizing the anti-ALP. A very efficient detection of very low ALP concentrations in PBS solution, ranging from 0.03 U/L to 0.3 U/L (1 nM–10 nM respectively), as well as a high selectivity due to the antibody/antigen interaction were demonstrated. The remarkable sensitivity and selectivity achieved through the combination of SiNW-forest combined electrochemical impedance spectroscopy suggest an effective strategy for real time and quantitative sensing of ALP.
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An inhibition type alkaline phosphatase biosensor for amperometric determination of caffeine

Abstract

Introduction

Section snippets

Chemicals

Apparatus

Determination of the inhibition effect of caffeine on ALP

Conclusion

Phytomedicine

Anal. Chim. Acta

Food Chem.

Anal. Biochem.

Talanta

Anal. Chim. Acta

Food Chem.

J. Food Comp. Anal.

Talanta

J. Chromatogr. A

Anal. Chim. Acta

J. Food Comp. Anal.

Food Chem

J. Pharm. Biomed. Anal.

Food Chem.

Microchem. J.

Anal. Chim. Acta

Clin. Chim. Acta

Electrochim. Acta