Development of an ion-channel sensor for heparin detection
Introduction
Since the introduction of ion-channel or ion-channel–mimetic sensors in 1987 [1], research efforts have been directed at the use of this technique for the detection of various ions and molecules [2]. For example, they have been used for the detection of hydrogen ions [3], [4], [5], [6], metal cations [1], [7], [8], [9], [10], nucleotides [11], [12], [13], [14], inorganic anions [15], antibodies [16], [17], and dopamine [18]. The principle of this technique is based on binding of the analytes to receptors at an electrode surface, which controls the reduction or oxidation of electroactive ions or molecules, often referred to as markers. This control is either due to physical exclusion [1] or electrostatic attraction or repulsion [1], [11], [12] between the receptor/analyte complexes and the electroactive markers. Because the working principle of these sensors is similar to that of ion-channel proteins in biomembranes, these sensors have been called ‘ion-channel sensors’ or ‘ion-channel–mimetic sensors’ [2], [19], [20]. Ion-channel sensors offer the advantages of robustness, ease of electrode preparation and regeneration, relatively fast analysis time, ease of automation and reasonable instrumentation cost. These advantages are paramount for techniques in routine analysis. Recently, we have reported that an ion-channel sensor based on electrodes chemically modified with self-assembled monolayers (SAMs) of thioctic acid can detect protamine, a polycation, at concentrations as low as 0.5 μg/ml when [Ru(NH3)6]3+ is used as a marker ion [21]. The detection of polyions with ion-channel sensors is advantageous since the large charge numbers on polyions (about −70 for heparin) result in strong binding to receptors and, therefore, allow highly sensitive detection of the polyions. The detection of protamine with an ion-channel sensor has encouraged us to further investigate the use of ion-channel sensors for the detection of heparin, a polyanion and an important biopolymer.
Heparin is extensively used as an anticoagulant in many clinical procedures for the prevention of blood clotting, especially during open heart surgery [22], [23]. The normal heparin concentration during these procedures is between 2 and 8 U/ml, which corresponds to 0.8–3.2 μM or 12–48 μg/ml [24]. In the treatment of post-operative thrombosis and embolism, however, the therapeutic concentration range of heparin is 0.2–0.7 U/ml [25]. This implies that any method for heparin analysis should be able to detect heparin at concentrations as low as 0.2 U/ml. Heparin has a molecular weight ranging from 5,000 to 30,000 with an average molecular weight of 15,000. At physiological pH, heparin is ionized and becomes negatively charged due to complete ionization of sulphate (ROSO3− and RNHSO3−) and carboxylate (RCOO−) groups [26]. The method commonly used for heparin determination in blood samples is the activated clotting time (ACT) method or the activated partial thromboplastin time (APTT) method [27]. In these methods, the clotting time of the plasma sample is measured after an initiator of the clotting process has been added. The heparin activity is then determined from the delay in the appearance of a clot. Typical clotting times for normal plasma are in the order of 30–40 s. Even though these methods give accurate results and have been in use for quite a long time, it has been reported that the clotting times determined by these methods not only depend on the amount of heparin but also on the concentration of antithrombin III (ATIII) and other coagulation factors [28].
Research efforts have been directed at developing alternative methods of heparin analysis in recent times. A heparin responsive potentiometric sensor has been reported with a detection range of 1.0–9.8 U/ml [29], [30], [31], [32], [33], [34], [35], [36]. This sensor employed tridodecylmethylammonium chloride (TDDMACl) as a sensing element incorporated in a polymeric liquid membrane. This polymeric membrane electrode has been used successfully to determine heparin concentrations in blood samples. van Kerkhof et al. have reported on a heparin sensor based on protamine as affinity ligand using an ion-sensitive field effect transistor (ISFET) [28], [36]. An indirect ion-step method was employed in this technique to detect heparin. The detection range of this sensor was reported to be 0.1–2.0 U/ml. A surface plasmon resonance sensor based on protamine and polyethylene imine (PEI) as affinity ligand has also been reported for heparin measurements in blood plasma with a detection limit of 0.2 U/ml and a linear range of 0.2–2.0 U/ml [37], [38], [39].
In this paper we report the detection of heparin using ion-channel sensors based on electrodes chemically modified with SAMs. The sensor has been applied to the measurement of heparin in artificial and horse serum.
Section snippets
Reagents
Thioctic acid (1,2-dithiolane-3-pentanoic acid), N,N′-dicyclohexyl carbodiimide, and protamine sulphate (salmine sulphate from salmon sperm with average Mr of 4,500) were obtained from Tokyo Chemical Industry (TCI), Tokyo, Japan. [Ru(NH3)6]Cl3 and (2-aminoethyl)trimethyl ammonium chloride were purchased from Aldrich, Milwaukee, WI, USA, and triethylamine from Kokusan Chemical Works, Tokyo, Japan. Tris(hydroxymethyl)aminomethane (Tris), N-hydroxysuccinimide, albumin (bovine serum, low salt), K3
Thioctic acid SAMs on gold electrode
Electrodes chemically modified with SAMs of thioctic acid have been reported as ion-channel sensors for the detection of protamine [21]. The negative charges on the surface of self-assembled thioctic acid monolayers caused protamine to bind electrostatically to the thioctic acid monolayers. This allowed negative marker ions in solution to approach the electrode surfaces or repulsed positive marker ions from them, thereby controlling the redox current of the marker ion. The protamine bound to
Conclusions
Ion-channel sensors have been used for the detection of heparin with [Mo(CN)8]4− or [Fe(CN)6]3− as electroactive markers. The detection is based on the attachment of protamine onto self-assembled monolayers of thioctic acid. The sensors have been used to determine heparin in both artificial and horse serum. Preliminary results with a quaternary ammonium receptor for heparin suggest that further improvements in detection limits and ease of electrode regeneration can be obtained by use of
Acknowledgements
Y.U. acknowledges the Japan Society for the Promotion of Science (JSPS) for the Invitation Fellowship Programme for Research in Japan (long term) to V.P.Y.G. A JSPS fellowship to K.P.X. is also gratefully acknowledged. We thank Daiichi Chemical Company, Tokyo, Japan and Tissue Culture Biologicals, Tokyo, Japan for the kind donation of heparin and horse serum, respectively. This work was supported by Grant-in-Aid for Scientific Research for the Priority Areas of ‘Electrochemistry of Ordered
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- 1
On sabbatical leave from the Department of Chemistry, University of Cape Coast, Ghana.
- 2
Research Fellow of the Japan Society for the Promotion of Science (JSPS). Present address: Department of Chemistry, Michigan State University, East Lansing, USA.