Evaluation of Acetylcholine Release and Hold Electrochemical Device by CCD Type Ion Image Sensor
Authors: Kageyama Itsuki , Kato Ryo , Sawada Kazuaki , Hattori Toshiaki 
Published: February 2020

Introduction Acetylcholine (ACh) is well known as one of the important neurotransmitters. Neurotransmitters accomplish neural communication in a synapse which is a small gap between neurons. Imaging the dynamics of neurotransmitters is essential for the total understanding of neuronal communication. We have been developing a semiconductor array ion image sensor using CMOS and CCD technologies to observe cells and tissue by use of ions1). However, the biological observation of living cells requires also developments of tools to manipulate cells and to add stimuli. There are several types of stimulation method, such as electrical, physical, optical and chemical stimulations. Chemical stimulation method has advantage that the dynamics is understood clearly, because a specific receptor responds to a specific stimulant. There are some problems in the general chemical stimulation method. In the case of injection method of stimulant solution, it is difficult to control of stimulation area. In the case of stimuli using caged compounds and photoirradiation, time-consuming pretreatment is required, and it must be considered inactivation and damage of cells by caged compounds. Hence, the development of local chemical stimulation method without pretreatment is desired. In this study, we developed an ACh release and hold electrochemical device. This device consists of three layers; PVC membrane including ACh and tetrakis[3,5-bis(trifluoromethyl)pheny]borate (TFPB), PEDOT layer and gold electrode. This device can release ACh by control of voltage according to the following principle. When a voltage is applied to the device, conductive polymer PEDOT is oxidized and charged positively, and TFPB anion is doped from PVC membrane. Then, ACh in the PVC membrane is released into the sample solution in order to maintain neutrality of charge in PVC membrane.

Experimental A gold disk electrode (3 mm diameter) was immersed in acetonitrile containing EDOT and tetrabutylammonium perchlorate, and was modified with PEDOT layer by cyclic voltammetry (CV)2). The PEDOT electrode was coated with plasticized PVC membrane containing ACh-TFPB complex by conventional cast method. The ACh-TFPB ion-pair was prepared by evaporation of a dichloromethane after mixing AChCl aqueous solution and NaTFPB dichloromethane solution. Characteristics of the device were evaluated by CV. ACh released from the device was detected using two ion image sensors; ACh image sensor3) and lipophilic cation image sensor4). The ACh image sensor is constructed by immobilizing acetylcholinesterase (AChE) in polyion complex membrane on a pH image sensor. ACh is hydrolyzed to choline and acetic acid by AChE. ACh is observed by detecting pH change by enzyme reaction. The lipophilic cation image sensor is constructed by coating lipophilic cation selective plasticized PVC membrane on a CCD type ion image sensor. This sensor detects membrane potential to lipophilic cation such as ACh. After ACh was released from the device, negative potential was applied to the device in AChCl solution in order to re-charge ACh to the device. And then ACh release was monitored again using lipophilic cation image sensor.

Results and discussion As a result of CV in tris-buffer solution (pH 7.1), it was found that oxidation of PEDOT was observed at about 0.8 V. When a potential 1.0 V was applied to the device on the ACh image sensor, decrease in local pH was observed at just-under position of the device. It is confirmed that ACh was released from the device by application of the voltage, while the detection of ACh was slow. However, the lipophilic cation image sensor monitored a faster releasing of ACh than the ACh image sensor. The results of both ion image sensors indicated the device released ACh locally and immidiatedly after the application of the voltage. After the re-charge of the device, the local release of ACh was also observed. The device could be used repeatedly at least 9 times.
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