FLOW CELLCholinesterase activity

The determination of serum cholinesterase activity and of the dibucaine number plays an important role in anaesthesiology. This originates from the early 1950s when a new short-acting muscle relaxant, succinylcholine,was introduced as an inhibitor of the motor receptors in subjects under anaesthetics. In normal subjects this effect is usually short, but if the cholinesterase activity is low or genetically altered, the patients may display prolonged apnoea. Since succinylcholine can only be hydrolysed by blood cholinesterase, it becomes of fundamental importance to evaluate the cholinesterase activity in the blood as well as the dibucaine number, defined as the degree of cholinesterase activity inhibition caused by a 10micromol/litre concentration of dibucaine with benzoylcholine as substrate (Garry, 1971).

This test clearly identifies the ‘typical’, ‘atypical’ and heterozygous forms of serum cholinesterase, their dibucaine inhibitions being about 80%, 20% and 40-70% respectively. Failure of the ‘atypical’ enzyme to hydrolyse succinylcholine may result in prolonged apnoea.

Numerous methods to determine the activity of serum cholinesterase and the dibucaine numbers have been described (Kalow &Genest, 1957; Evans & Wroe, 1978; Okabe et al.,1980; Whittaker et al.,1983; Panteghini et al., 1986). These methods, based mainly on spectrophotometric measurements, often have disadvantages such as interferences, long procedure time and expensive instrumentation. Methods based on pH and differential pH measurements have also been proposed (Witter et al., 1966; Barenghi et al.,1986), as methods based on the use of oxygen and hydrogen peroxide sensors (Mizutani &Tsuda, 1982; Yao, 1983). Although these methods offer good accuracy and precision, they lack in sensitivity and require modified silica gel columns to remove interferences such as ascorbate, urate, tyrosine, cysteine, glutathione and bilirubin.

A hydrogen peroxide electrode assembled as a choline electrode was used, which was unaffected by the above interferences. It allowed the determination of cholinesterase activity in 2 min and the dibucaine number in 5 min, with a precision of 2%, allowing clear identification of the three forms of this enzyme.


Cholinestrase activity for detection of organophosphorous and carbamic pesticides in environmental and food samples

Biosensors have known a large development in analytical determination of biological and organic analytes. The use of biosensors permits to obtain a quick, sensitive and specific electric signal strictly related to analyte concentration in real samples.

Many efforts have been done in pesticides analytical determination. Organochlorine insecticides (e.g. DDT, aldrin, lindane) were progressively replaced by organophosphorus (e.g. parathion, malathion) and derivatives of carbamic acid (e.g. carbaryl, aldicarb) insecticides, that show lower persistence in the environment but represent a serious risk because of their high acute toxicity. In fact, they are inhibitors of the cholinesterase enzymes, involved in muscle contraction and impulse transmission in nervous system. Main risks regard both the professional (industrial production, agricultural use) and not professional exposures (domestic use, food and fresh water contamination).

Several organophosphorous (OP) compounds are still used as insecticides and chemical warfare agents. The most frequently used methods for detection of OP and CB pesticides are based on gas and liquid chromatography in combination with mass spectrometry. While being sensitive and reliable, such methods require expensive laboratory equipments, trained personnel and complex procedures to treat and extract the real matrices. These procedures allow to discriminate between different compounds which belong to the same class, but, in spite of their sensitivity, these methods are time consuming and need specialised analytical laboratory. Furthermore these methods do not provide any information about anti cholinesterase activity (toxicity) of these compounds.

The use of biosensors as an analytical method to determine the presence of neurotoxic pesticides represents an attractive alternative for research because of their high sensitivity, selectiveness, ease and rapidity of use. These analytical devices, based on the intimate contact between a biorecognition element that interacts with the analyte of interest and a transducer element that converts the biorecognition event into a measurable signal, are gaining momentum as complimentary screening assays together with classical analytical techniques due to their high selectivity and sensitivity, low instrumentation cost, easier procedures and rapidity of the assay. After the rapid screening step performed with biosensors on many real samples, only the positive ones can be performed with the classical analytical techniques with the aim of lowering the cost of field analysis.

Among the different types of biosensors, the electrochemical ones are especially interesting due to the high sensitivity inherent to the electrochemical detection and the possibility to miniaturise the required instrumentation, providing compact and portable analysis devices. Electrochemical, mono or bi-enzyme, biosensors able to recognize CBs and OPs by specific inhibition of acetyl cholinesterase (AChE) as biorecognition element have been extensively reported (Cremisini, Di Sario, Mela, Pilloton, & Palleschi, 1995; Hernandez, Palchetti, & Mascini, 2000; Albareda-Sirvent, Merkoçi, & Alegret, 2001; Andrescu, Barthelmebs, & Marty, 2002; Andreescu, Avramescu, Bala, Magearuy, & Marty, 2002; Vastarella, Rosa, Cremisini, Della Seta, Montereali, & Pilloton, 2007). In both categories OPs and CBs determination is performed by AChE activity measurements before and after exposure to a sample (incubation) and calculation of the inhibition due to OP and CB compounds. Performance of bi-enzymatic biosensor was based on two enzymatic reaction in series as reported in (1) and (2). Acethylcholine + H2O → acetate + choline (1) Choline +2 O2 → betaine aldehyde + 2 H2O2 (2) OP and CB lower the enzyme activity of the first enzyme and therefore the production of choline and subsequently reduce the current produced by oxidation of hydrogen peroxide.

In mono enzyme biosensor, a modified substrate acetylthiocholine (ATCh) was used. In this system the specific inhibition of AChE by pesticides gives rise to a minor production of thiocholine which can be directly oxidised on the electrode surface and so no additional enzyme is requested to detect the lower current intensity due to inhibition of AcChE.

In addition the enzyme (AChE) used in this biosensor is the same which determines neurotoxic effects in humans and animals and, for this reason, the measurements are directly related to the biological effect while the traditional chromatographic methods detect the concentration of the pesticides without any information about the neurotixicity due to the mix of different compounds. For inhibition biosensors, the calibration curves and the measurements on real samples are conventionally expressed in concentration units of a pesticide which gives the same neurotoxic effect. Research and development on biosensors for pesticides had many improvements in the last two decades and are still going for ever increasing complexity of the immobilization strategies of the enzymes (Viswanathan, Radecka, Radecki, 2009), the use of novel biological mediators as well as bacteria (Stoytcheva, Zlatev, Velkova, Valdez, Ovalle, Petkov, 2009) and miniaturization of the transducers (Guerrieri, Monaci, Quinto and Palmisano, 2002). Some authors (Bachmann & Schmid, 1999; Cortina, Del Valle, & Marty, 2008) proposed disposable screen-printed amperometric multielectrode biosensors based on inhibition of different types of native and recombinant AChEs and chemometric data analysis using Artificial Neural Networks (ANNs). Nevertheless these advanced studies the applications to food industry requirements and real samples remain poor and not much explored, especially for screening food samples.

The pesticide determination is performed with a kinetic measurement of the initial velocity of the reaction catalysed by this enzyme before and after an incubation step with pesticides in the real sample. The kinetic measurement has been performed by using different electrochemical transducers as glass electrodes with differential pH meter, ISFETs, Light Addressable Potentiometric Sensor (LAPS), conductivity cells, amperometric carbon modified electrode or with an amperometric choline oxidase based biosensor. pH and conductivity measurements suffer of reproducibility and low sensitivity and the use of new generation carbon modified electrodes requires frequent polishing of sensor surface. Many biosensors have been developed for in situ determination of OP compounds, especially those based on amperometric detection of the AChE activity, as supporting tool of the classical analytical techniques.

Single use or disposable biosensors based on thick film technology, are attractive in on-field measurements wherein irreversible processes take place such as in this case. Screen printed electrodes (SPEs) are particularly suitable in low cost, easy to be used instrumentation, even compatible with hand-held analysers. The procedure to determine the acetyl cholinesterase inhibition, using diethyl p-nitrophenyl phosphate (paraoxon) as a reference pesticide is based on a double-enzymatic amperometric sensor by measuring the re-oxidation current of hydrogen peroxide generated as a final product of the following enzymatic sequence:

a) AChE catalyzed hydrolysis of acetylcoline to choline;

b) oxidation of choline to betaine by choline oxidase (ChOx).

Many authors have used AChE free or immobilised on the electrode surface. In all cases measurement of AChE inhibition has been performed directly in the sample or in a diluted solution of the sample. With this approach, the determination could be affected by electrochemical interferences in the sample altering the sensor performances.

In our procedure AChE was separately immobilised on a nylon membrane for the measurement of residual cholinesterase activity after inhibition. Effective measurement of the residual enzyme activity was therefore performed after the incubation in a standardized solution with a ChOx electrochemical biosensor. Homemade SPE were used for amperometric detection. Because the detection of the AChE activity was performed in a standard solution of the substrate, the proposed method is unaffected by interferences, avoiding the use of protecting selective membranes which greatly affect the sensitivity of biosensors. Incubation simultaneously performed on a number of samples enabled to lower the analysis time per sample. High sensitivity and reproducibility were then obtained for the analysis of paraoxon.

  • OP and CB in strawberries

The procedure was extended to the determination of cholinesterase inhibitors in strawberries, adopting a very simple preparation of fruit sample, at the beginning of the above described analytical sequence. These samples were tested to verify any residual presence of cholinesterase inhibitor.

  • OP and CB in EVO

Extra Virgin olive oil (EV) is one of the most known and typical food products of the Mediterranean regions and Spain, Italy and Greece represents the main EV producers in the world. Its popularity depends on its appreciated sensorial properties besides positive effects for human health. EV total quality depends on multiple factors such as olive cultivars, production process, preservation methods and, last but not least, agronomic practices.

Organoposphourus (OP) and Carbamic (CB) pesticides are normally used in olive fruit production to keep crops healthy and to prevent them being destroyed as a consequence of disease and infestation. A possible consequence of their use may be the presence of pesticide residues in the EV extracted from treated fruits. Maximum residue levels have been established by different institution (Food and Drug Administration, Codex Alimentarius Commission) in order to avoid possible risks for human health (ECs no 396/2005; n°178/2006 and n° 149/2008). A recent research (Dominici, Guadagnini, Sciarra & Ucci, 2008) about pesticides contamination in commercial Italian EV olive oils shows 1.9% of irregular samples, 3,4% of regular samples with only one class of residues and 12% of regular olive oil samples with more than one residue, thus confirming the importance of the development of multi-residue analytical methods for the determination of pesticides in this important food product.

The analysis of pesticide residues in EV is complicated, because of the inherent complexity of the matrix, mainly comprising triglycerides (98–99%). Many pesticides are fat-soluble non-polar compounds and tend to concentrate and remain in the fat matrix. This behavior normally requires the use of additional extraction clean-up steps, to isolate or to extract the pesticide fraction from the whole fatty matrix, prior to conventional (Gilbert-López, García-Reyes, & Molina-Díaz, 2009) or biosensor analysis (Del Carlo, Mascini, Pepe, Diletti, & Compagnoni, 2004).

The main strategies used for the isolation of pesticides in olive oil samples involve the application of techniques called liquid-phase extraction (LPE) or liquid–solid extraction (LSE). In both cases the use of organic solvents such as hexan, acetonitrile was required (Gilbert-López, et al., 2009). These remarks highlight the advantage to develop pesticide biosensor working in organic solvent able to analyze food samples with relatively high fat content (i.e. >15%), such as olive oil. Previous researches (Mionetto, Marty, & Karube, 1994; Palchetti, Cagnini, Del Carlo, Coppi, Mascini, & Turner, 1997; Campanella, De Luca, Sammartino, & Tomassetti, 1999; Andreescu, Avramescu, et al., 2002) have shown the capability of acetylcholinesterase-choline oxidase bi-enzymatic biosensor working in non aqueous solvents to obtain small or no inhibition effect concerning acetylcholinesterase activity in different organic solvents.

An important goal which could be obtained with amperometric biosensors based on inhibition of AChE is the capability to distinguish between pesticide classes (OP and CB) when both are present in a mixture.

We used a basically assembled biosensor as extensively reported in literature, for the development of a simple, inexpensive, and sensitive screening method based on bi-enzimatic (AChE–ChOX) amperometric biosensor able to obtain quantitative information with regard to the simultaneous presence of OPs and CBs in olive oil samples.

The recognition of the two different pesticide classes by AChE–ChOX biosensor has been carried out by exploiting the well known different inhibition mechanism of CB and OP compounds versus AChE activity. CB compounds form an unstable bond with esterasic site of AChE that spontaneously tends to break down in an interval time ranging from few minutes to one hour (reversible inhibition). OP compounds, on the contrary, form a highly stable covalent bond phosphate-enzyme, bringing about an irreversible inhibition (Fukuto, 1990).

Inhibition tests on AChE were carried out with OPs and CBs solutions at different concentration in hexan, whereas the tests on real samples were straight followed on spiked EV sample diluted with hexan solvent without no extraction step. To improve the speed and reproducibility of the analysis, the bi-enzimatic biosensor was connected to a Flow Injection Analysis (FIA) manifold.

  1. Quantitative screening and resolution of carbamic and organophosphate pesticides mixture in extra virgin olive oil by acetylcholinesterase -choline oxidase sensor - D. Albanese , M. Di Matteo and R. Pilloton - Journal of Environmental Science and Engineering A 1 (2012) 68-77
  2. A preliminary study on electrochemical biosensors for the determination of total cholinesterase inhibitors in strawberries - W Vastarella, V Rosa, C Cremisini, LD Seta, MR Montereali, R Pilloton International Journal of Environmental and Analytical Chemistry 87 (10-11) 2007, 689-69
  3. Evaluation of the use of free and immobilised acetylcholinesterase for paraoxon detection with an amperometric choline oxidase based biosensor C Cremisini, S Di Sario, J Mela, R Pilloton, G Palleschi Analytica chimica acta (1995) 311 (3), 273-280
  4. Optimised Biosensors Based on Purified Enzymes and Engineered Yeasts: Detection of Inhibitors of Cholinesterases on Grapes - A. Boni, C. Cremisini, E. Magaro`, M. Tosi, W. Vastarella,and R. Pilloton - ANALYTICAL LETTERS Vol. 37, No. 8, pp. 1–16, 2004
  5. Determination of serum cholinesterase activity and dibucaine number by an amperometric choline sensor - G Palleschi, MG Lavagnini, D Moscone, R Pilloton, D D'Ottavio, ME Evangelisti Biosensors and Bioelectronics (1990) 5 (1), 27

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