DOI: 10.1002/elan.201900507 AcetylcholinesteraseSensorBasedonPolyelectrolyte ComplexeswithDNAinclusionfortheDeterminationof Reversible Inhibitors R. Davletshina, [a] A. Ivanov, [a] and G. Evtugyn* [a] Abstract: The acetylcholinesterase (AChE) biosensor has been developed for the determination of reversible inhibitors applied in the Alzheimer’s disease therapy, i.e., Huperzine A (HupA) and galantamine (Gal). For this purpose, glassy carbon electrode (GCE) was first modi- fied with carbon black (CB) and Co phthalocyanine and then polyelectrolyte complex was self-assembled on its surface by drop casting of reactants and washing. To extend the stability and improve biosensor performance, it was proposed for the first time to use DNA as polyanion in the complex assembly. The DNA showed higher charge density than conventional polyelectrolytes and stabilized the surface coating by adsorption of higher enzyme amount and prevention of its leaching during the biosensor operation. Complex formation and the influ- ence of structural factors were monitored with surface plasmon resonance. Kinetic study showed mixed inhib- ition of the enzyme within micro- and nanomolar range of inhibitor concentrations. The AChE biosensor showed limit of detection of HupA equal to 0.9 and that of Gal to 70 nM. The sensitivity of drug determination was found to be close or better than that of the AChE biosensors previously reported in the literature. The biosensor was tested on the sample of artificial urine and showed 102% recovery of the drugs determination. Keywords: acetylcholinesterase biosensor · reversible inhibition · polyelectrolyte complex · Alzheimer’s disease drug 1Introduction Recently, an interest to the AChE analytical application has been enormously increased due to high attention devoted to neurodegenerative diseases. Thus, Alzheimer’s disease is attended with suppression of activity of choline acetyltransferase which controls synthesis of acetylcho- line. Application of the AChE reversible inhibitors compensates for the decay of acetylcholine concentration and hence suppresses the symptomatology of the disease [1,2]. In this respect, there is an urgent need in development of fast and sensitive sensors for the determination of reversible AChE inhibitors and control of the doze of medications to avoid occasional poisoning of the patients by exhausting drug usage. Although biosensors with immobilized AChE are well known [3–6], they are mostly intended to the determination of irreversible inhibitors, i.e., chemical warfare and organophosphate pesticides. Meanwhile the assay of reversible inhibition offers stricter requirements to the immobilization conditions because enzyme-inhibitor interaction is conducted in the presence of a substrate and reaction conditions affect the sensitivity of enzyme active sites toward analytes in a more complicated manner. In this work, non-covalent immobilization of the AChE in polyelectrolyte layers was investigated and working conditions for detection of model reversible AChE inhibitors, i.e., ()-huperzine A (HupA) and galantamine (Gal) were specified to reach maximal sensitivity of their determination. Chemical structures of the inhibitors are presented in Figure 1. In China, alkaloid HupA extracted from Huperzia serrata is approved as drug in therapy of Alzheimer’s disease after IV phase of clinical trials [7]. In the USA, HupA was allowed for use as a food additive [8]. In [a] R. Davletshina, A. Ivanov, G. Evtugyn A. M. Butlerov’ Chemistry Institute of Kazan Federal Uni- versity, 18 Kremlevskaya street, Kazan 420008, Russian Fed- eration E-mail: Gennady.Evtugyn@kpfu.ru Supporting information for this article is available on the WWW under https://doi.org/10.1002/elan.201900507 Fig. 1. Chemical structures of the AChE inhibitors. Full Paper www.electroanalysis.wiley-vch.de © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2019, 31, 1 – 10 1 These are not the final page numbers! ��