Novel electrochemical method for sensitive determination of homocysteine with carbon nanotube-based electrodes

Abstract

An electrochemical method has been successfully demonstrated for sensitive determination of homocysteine (HcySH) with carbon nanotube (CNT)-modified glassy carbon (GC) electrodes. Cyclic voltammetric results clearly show that carbon nanotubes, especially those pretreated with nitric acid, possess an excellent electrocatalytic activity toward the oxidation of HcySH at a low potential (0.0 V versus Ag/AgCl). The remarkable catalytic property of the acid-pretreated CNTs, which is essentially associated with oxygen-containing moieties introduced on the tube surface, has been further exploited as a sensitive determination scheme for HcySH. Continuous-flow amperometric results suggest that the CNT-based electrodes (p-CNT/Nafion/GC), which were prepared by using Nafion to solubilize and further immobilize CNTs on GC electrodes, show striking analytical properties of good stability and reproducibility and strong ability against electrode fouling. Such analytical properties, along with the low operation potential, substantially enable a reliable and sensitive determination of HcySH with a good dynamic linearity up to 60 μM and a detection limit of 0.06 μM (S/N=3). The catalytic mechanism and the possible application of the as-prepared p-CNT/Nafion/GC electrodes for the study of the auto-oxidation of HcySH are also demonstrated and discussed.

Introduction

The development of a simple assay for the reliable and durable determination of homocysteine (HcySH) is of great physiological and pathological importance, because accumulating data from epidemiological studies have suggested that individuals with elevated blood levels of HcySH have increases in risks of cardiovascular disease and that even moderately elevated HcySH levels are associated with heart attacks, strokes, problems in pregnancy, and weakened cognition in middle and old age (Robinson, 2000, Ueland et al., 1993, Groote et al., 1996). In biological environments, HcySH is formed when methionine is converted to cysteine. Once formed, it may either be irreversibly metabolized by the transsulfuration pathway to cysteine or remethylated to methionine. Deficiencies of the enzymes involved in these pathways and/or cofactors necessary for the metabolism of HcySH can result in aberrant intracellular processing of HcySH, leading to hyperhomocysteinemia (Robinson, 2000, Ueland et al., 1993). In humans, the plasma HcySH level is regulated and the normal basal concentration ranks from 5 to 15 μM. For upper levels, hyperhomocysteinemia is described; an increment in fasting plasma HcySH of 1 μM has been associated with an increase in risk of coronary heart disease of 10–20% (Still and McDowell, 1998).

Considerable efforts have been paid on the determination of HcySH and several comprehensive reviews have appeared in literature (Still and McDowell, 1998, Ducros et al., 2002, D’Angelo and Selhub, 1997, White et al., 2002). As demonstrated, the determination of HcySH could be performed by immunoassay (Shipchandler et al., 1995), gas chromatography–mass spectroscopy (GC/MS) (MacCoss et al., 1999), HPLC (or capillary electrophoresis) with fluorescent (Okabe et al., 2002), laser-induced fluorescent (Bayle et al., 2002), mass spectrometric (Nelson et al., 2003), and electrochemical detectors (Inoue and Kirchhoff, 2002). Electrochemical methods are known to possess such advantages as simplicity, high sensitivity and ease in automation. However, such methods are greatly limited for accomplishing the above purpose because of the poor electrochemical behavior of HcySH at most commonly used electrodes, e.g., glassy carbon and gold electrodes. Although some improvements in the electrochemical responses of HcySH have been recently sought by the uses of diamond electrodes (Spataru et al., 2001, Terashima et al., 2003) and modified electrodes (Zen et al., 2001, Mao and Yamamoto, 2000), the potential of electrochemical methods used for the routine analysis of HcySH still remains to be exploited.

On the other hand, with novel structural, electronic, physical and mechanical properties, carbon nanotubes (CNTs) constitute an important new form of carbon materials that are finding striking applications in many fields (Baughman et al., 2002), such as energy conversion and storage (Che et al., 1998), electromechanical actuators (Baughman et al., 1999) and chemical sensing (Kong et al., 2000). Recent electrochemical studies have demonstrated that CNTs possess excellent electrochemical properties with a strong ability against electrode fouling (Nugent et al., 2001, Luo et al., 2001, Wu et al., 2003, Wang et al., 2003), suggesting their promising prospects in the development of electrochemical methodologies for biological-oriented determinations. In the present work we first demonstrate the striking catalytic activity of CNTs toward the oxidation of HcySH and, based on this, develop a novel electrochemical method for the durable and sensitive determination of HcySH. The possible application of the as-developed electrochemical method for the preliminary study of the auto-oxidation of HcySH is also described.