Amperometric acetylcholinesterase biosensor for physostigmine determination

R.R. Davletshina^*, A.N. Ivanov^**, G.A. Evtugyn^***

Kazan Federal University, Kazan, 420008 Russia

E-mail: ^*luckydav@mail.ru, ^**Alexey.Ivanov@kpfu.ru, ^***Gennady.Evtugyn@kpfu.ru

Received November 7, 2017

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Abstract

A biosensor based on acetylcholinesterase immobilized by carbodiimide binding on glassy carbon electrode modified with carbon black and Co(II) phthalocyanine has been developed. The working conditions for assembling of the surface layer and measurement protocol have been specified for mediated oxidation of the product formed in the enzymatic hydrolysis of acetylthiocholine. It has been demonstrated that immobilization of the enzyme does not decrease the affinity of the active site to the substrate. The possibility for determination of physostigmine as a model reversible inhibitor in the concentration range from 0.3 nM to 10 μM and the detection limit of 1.8?10^–10 M has been established. The kinetic parameters of enzymatic hydrolysis of the substrate and those of competitive inhibition of the enzyme have been calculated. The developed biosensor can find application for the monitoring of anticholinesterase drugs.

Keywords: acetylcholinesterase, enzyme inhibitor, biosensor, determination of reversible inhibition, cobalt(II) phthalocyanine, chemically modified electrode

Acknowledgments. The study was supported by the Russian Foundation for Basic Research (project no. 17-03-00381).

Figure Captions

Fig. 1. Dependence of the inhibition degree on the time of biosensor incubation in physostigmine solution. Physostigmine concentration – 0.01 μM, pH of physostigmine solution – 7.8, AChE concentration on the biosensor surface – 0.0024 AU/mm².

Fig. 2. Dependence of the inhibition degree on the pH value of physostigmine solution. Physostigmine concentration – 0.1 μM, incubation time – 15 min, AChE concentration on the biosensor surface – 0.0024 AU/mm².

Fig. 3. Dependence of the inhibition degree and the anode current of thiocholine oxidation on the amount of the enzyme used for AChE immobilization. Physostigmine concentration – 0.1 μM, рН 7.0, incubation time – 15 min, ATC – 5 mM. Mean values and measurement errors for six biosensors are provided.

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For citation: Davletshina R.R., Ivanov A.N., Evtugyn G.A. Amperometric acetylcholinesterase biosensor for physostigmine determination. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2018, vol. 160, no. 1, pp. 5–16. (In Russian)

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