Capillary electrophoresis of adenosine phosphates using boron-doped diamond electrodes

A capillary electrophoresis coupled with electrochemical detection using boron-doped diamond electrode was developed for simultaneous detection of adenosine phosphates, i.e. adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP). In phosphate buffer solution pH 7, these three adenosine phosphates have similar oxidation potentials at around +0.9 V (vs. Ag/AgCl), which indicated that the oxidation occurred at the same moiety. Capillary electrophoresis, which was then performed using fused silica capillary (dia. 0.05 mm) at an applied potential of 10 KV can separate ATP, ADP and AMP with the retention times of 848 s, 1202 s, and 1439 s, respectively. Linear calibration curves with the limits of detection of 0.59 μM, 0.56 μM and 1.78 μM, respectively, can be achieved, suggested that capillary electrophoresis with electrochemical detector is promising for simultaneous detection of adenosine phosphates.


Introduction
Adenosine phosphate is a nucleotide composed of pentose, adenine bases (purine), and phosphates. Adenosine phosphates have a various number of phosphates, that are adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP). Among these three types of adenosine phosphates, only ATP is used as a medical treatment for amyotrophia disease, cerebral hemorrhage and hepatitis [1]. Accordingly, a sensitive method to measure ATP in the mixture of those adenosine phosphates is necessary. Generally, HPLC is employed to determine the adenosine phosphates. However, HPLC is not a cheap instrument. A capillary electrophoresis method, which is performed based on the movement of the charged molecules through a fluid under an electric field influence, can be used as an alternative for the separation process of adenosine phosphates. Then, by combining the electrophoresis with a detector, the separated adenosine phosphates can be quantified.
The detectors commonly used in capillary electrophoresis are UV-visible and laser induced fluorescence [2]. UV-visible has the disadvantage in the detection limit, while the laser induced fluorescence is an expensive instrument. Meanwhile, boron-doped diamond (BDD) is established as an alternative of solid electrodes due to its superior properties among other electrodes, including its wide potential window, low background current, and high surface stability [3][4][5]. BDD have been investigated for electrochemical detection of adenosine phosphates [1], showing similar oxidation potentials at around +0.9 V for those three adenosine phosphates. In this work, BDD was employed as the detector for capillary electrophoresis of AMP, ADP, and ATP. Succesfull separation with good detection limits could be achieved, suggested that the method was promising for the real applications.

Materials and methods
Prior to use, the BDD film was cleaned subsequently by ultrasonication in 1-propanol and water for 15 min each. Cyclic voltammetry was performed in one-container electrochemical cell to study the electrochemical behaviour of adenosine phosphates in phosphate buffer solution (PBS). The capillary electrophoresis cell was self-fabricated and modified from the design of Shin et al. [2]. This cell was composed of a couple of platinum electrodes immersed in two separate containers. The electrodes were connected to a power supply to provide negative and positive poles, while between the containers, a silica fused capillary tube (i.d. 0.05 mm) was placed. A BDD film was arranged at the end of the capillary tube in the negative pole container and connected to a potensiostat with a Pt wire as the counter electrode and an Ag/Ag system as the reference electrode. The scheme of capillary electrophoresis cell is displayed in figure 1.

Results and discussion
Cyclic voltammograms (CVs) of 0.1 M PBS pH 7 in the absence and in the presence of AMP (figure 2a) in comparison with those of ADP and ATP ( figure 2b and figure 2c, respectively) at BDD electrode show an oxidation peak of all types of adenosine phosphates at the potential around +0.90 to +0.95 V (vs. Ag/AgCl). It can be seen that the more phosphate groups contained in the adenosine phosphates, the higher oxidation potential was required. However, the oxidation potential was observed in the narrow range between +0.90 to +0.95 V, suggested that the oxidation occurred at the same moieties. Adenine, the nitrogenous bases with electroactive properties, is the most possible moiety to undergo the oxidation reaction [1]. Furthermore, there was no reduction peak observed in the potential range between -1.0 to +1.5 V indicated that the oxidation reaction was irreversible.
Influence of the pH was also studied. Linear relationships were observed in the pH range between pH 2 and pH 8 for all adenosine phosphates with a slope of around -0.59 mV/pH (data not shown) suggested that the reaction involving the same numbers of proton and electron. Furthermore, linear calibration curves showed good linearity of the adenosine phosphates in the concentration range of 50 μm to 500 μm, indicated that the electrode can be applied for detection of all adenosine phosphates.   Capillary electrophoresis of the adenosine phosphates was performed at the separating potential of 10 KV. At the end of capillary tube in the negative pole container, BDD film was placed.