Assay of enzymes of clinical and biological significance by an interference free coline biosensor

Choline (Ch) is widely distributed in nature since it is an important source of methyl groups, an essential component of certain lipids and, in the nervous tissue of most organisms, a precursor of acetylcholine, which is a major neurotransmitter. Ch can be selectively determined by detection of H2O2 generated by the choline oxidase (ChO) catalyzed reaction. Several amperometric methods, based on the this reaction, have been developed in order to detect choline or species which determine choline release such as cholinesterase (ChE), acetylcholine (AChE), phospholipase D (PLD) and choline containing phospholipids. Measurement of serum ChE is important to assess liver function and monitor excessive exposure to organophosphorus insecticides[1]. It is also useful in predicting susceptibility to prolonged apnea after the administration of succinylcholine[2]. There are many methods for the measurement of cholinesterase activity including manometric, titrimetric and photometric procedures. The reference procedure for ChE assay is the Ellman colorimetric method[3] in which haemoglobin and glutathione present in erythrocytes act as interfering substances. PLD is widespread in plants as well as in some microorganisms and mammalian tissues and can have multiple effects and significance on cellular functions, such as receptor signal transduction[4] and an important role in postharvest metabolism of plant tissue. Methods published[5] for PLD activity assay have mainly involved the determination of choline by bromothymol blu or by synthetic substrates of PLD. Titrimetric or pH-stat techniques have been described as well as radioassay procedure which is the fastest and most sensitive method but it requires the use of expensive and potentially health hazardous radiolabeled phospholipids. The fundamental role covered by ChE and PLD in clinical and biological fields justifies the increasing interest in developing assay methodologies able to assure sensitivity, accuracy without requiring expensive instrumentation or long procedure time. In this context amperometric biosensors based on ChO play a surely innovative and quite promising role. The techniques reported for immobilizing ChO on the electrode surface are quite laborious. Covalent immobilization on nylon net[6], often coupled to multimembranes assembly[7] in order to preserve the electrode from interference and fouling, besides being complex and time consuming, can cause a certain slowing down of the sensor response. On the contrary a fast response time is an essential requirement for the on line monitoring of analytes in real samples. The aim of the present study was to develop a choline amperometric biosensor easier to realize moreover assuring high enzyme stability, fast response time, anti-interferent and anti-fouling properties. This goal has been reached in our laboratory immobilizing choline oxidase by co-crosslinking on a platinum electrode previously modified by an overoxidized polypyrrole film. Such an immobilization procedure, already reported in the case of a bienzymic sensor based on choline oxidase and acetylcholinesterase co-immobilization on a platinum electrode[8,9], allowed to obtain an immobilized enzyme-layer characterized by high biocomponent stability and good mechanical properties. Moreover the employment of a bilayer made of co-crosslinked choline oxidase and overoxidised polypyrrole assures notable permselectivity[10] allowing the rejection of interferents usually present in real matrices. Such a Ch biosensor, being interference free, has been employed to assay ChE and PLD in real matrices. In order to optimize the sensor response towards the enzyme to be assayed, the influence of experimental variables such as pH of buffer solution, rotation rate of the electrode and the substrate concentration has been studied. The present method, upon optimization, allowed wide linear range up to 0.600 UI/ml in the case of serum ChE (referred to acetylcholine as enzyme substrate) and to 0.33 UI/ml in the case of PLD. Moreover it revealed suitable for assay ChE in serum samples and PLD in plant crude extracts at activities value respectively down to 5x10-4 UI/ml and to 8x10-5 UI/ml. [1] E. Silk, J. King, M. Whittaker, Ann. Clin. Biochem. 16 (1979) 57 [2] A. Dietz, HM Rubinstein, T. Lubrano, Clin. Chem. 19 (1973) 1309 [3] G. L. Ellman et al. Biochem.Pharmacol. 7 (1961) 88-95 [4] J. H. Exton J. Biol. Chem. 272 (1997) 15579 [5] A. J. Morris, A. M. Frohman, J. Engebrecht, Analytical Biochemistry 252 (1997) 1 [6] E. Vrbovà, I. Kroupovà, O. Valentovà, Z. Novotnnà, J. Kas, Analytica Chimica Acta 280 (1993) 43 [7] G. Palleschi, M. Lavagnini, D. Moscone, R. Pilloton, D. D’Ottavio, M. E. Evangelisti Biosensors & Bioelectronics 5 (1990) 27 [8] A. Guerrieri, G.E. De Benedetto, F.Palmisano, P.G. Zambonin, Analyst 120 (1995) 2731 [9] A. Guerrieri, F. Palmisano, Anal. Chem. 73 (2001) 2875 [10] A. Guerrieri, G. E. De Benedetto, F. Palmisano, P. G. Zambonin, Biosensors & Bioelectronics 13 (1) (1998) 103