(Invited) Development of Bipolar Electrochemical Microscopy for High-Speed and High-Resolution Monitoring of Biochemical Phenomena
Authors: Inoue Kumi Y. , Kubota Koki , Endo Ayane , Iwama Tomoki , Ito-Sasaki Takahiro , Shiku Hitoshi 
Published: December 2024

Closed bipolar electrochemistry (cBPE) involves simultaneous oxidation and reduction at opposite poles of a conductor in two separated solutions. (Figure left). The unique property of cBPE to be operated without direct connection to an external power source is expected to lead to higher resolution in electrochemical imaging than the conventional microelectrode array type imaging platforms. We have applied cBPE to an electrochemical microscopy (BEM) (Figure right) for high-resolution biochemical imaging.1-
7 The operation principle of BEM has been successfully verified,1 then the current focus is on fabrication method of cBPE array. Previously, we filled Au into pores of a polyethylene terephthalate track-etched membrane having D=8 µm pores by electroless plating using 50 mM HAuCl4 aqueous solution and 50 mM NaBH4 EtOH solution.2 Using this cBPE array, an image of oxygen concentration distribution around cell spheroids was successfully obtained, but this method has a problem of the inability to completely fill the pores with gold, resulting in large ionic currents. In this study, we use conductive pastes for fabrication of vertical closed bipolar electrode array to completely fill the pores with conductive materials.
Epoxy-based carbon paste (ECP) was prepared by mixing acetylene carbon black (ACB; particle size=42 nm; Strem Chemicals) and epoxy resin (mixture of Epok 812 (62 mL), DDSA (100 mL), and DMP-30 (1.5%); Okenshoji). Oil-based carbon paste (OCP) was prepared by mixing the ACB and the liquid paraffin. For Au paste (AuP), commercially available AURoFUSE (particle size (D50) 0.2–0.4 µm; Tanaka Kikinzoku Kogyo) was used as is. Vertical cBPE arrays using the carbon paste filled in pores of a glass capillary plate was fabricated by filling ECP (ACB: epoxy resin=1:4 in weight ratio) cured at 60°C for 15 h. After cooling, the surface was coated by OCP (ACB: liquid paraffin=1:1 in weight ratio) to fill the dented parts shrank by heat curing. For [Fe(CN)6]3
− imaging, a sample side of the array was modified with reduced graphene oxide (RGO) by coating OCP containing 5 wt.% RGO (RGO-OCP). For H2O2 imaging, a sample side of the array was modified with Prussian blue (PB) by coating commercially available PB containing carbon paste (Carbon Mediator Paste, C2070424P2; SunChemical Gwent). The BEM system was constructed using two solution chambers (detection/sample chambers), BPE array, and driving electrodes. The detection chamber located at the bottom side of the BPE array was filled with 10 mM [Ru(bpy)3] Cl2/20 mM tripropylamine (TPA) solution containing phosphate buffered saline (PBS). The sample chamber located at the bottom side was filled with the sample solution for each measurement. A platinum driving electrode inserted into the detection chamber, another platinum driving electrode and a Ag/AgCl reference electrode inserted into the sample chamber were connected WE, CE and RE connector of a potentiostat, respectively. The entire system was mounted on an inverted microscope with a high sensitivity CCD camera to observe the electrochemiluminescence (ECL) generated at the underside of the BPEs.
By using RGO and PB modified carbon paste (PC) cBPE array for BEM element, we successfully obtained an image of 5 mM [Fe(CN)6]3− and H2O2 inflow, respectively.5 These results show that cBPE arrays for BEM can be fabricated by filling micropores with carbon paste taking advantage of the flexibility and ease of modification of the paste electrode. We are now investigating the use of AuP as an electrode material of cBPE array. We have obtained the result that ECL can be enhanced by using AuP as an electrode material.7 This may be due to the surface plasmon enhancement effect by the Au particles contained in the AuP. The establishment of a simple method for fabricating vertical cBPE arrays without ion leakage is expected to further accelerate the research and development of BEM.
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Iwama, M. Komatsu, K. Y. Inoue, K. Kubota, T. Ito-Sasaki, H. Shiku, SSRN, posted 28 Nov 2023, available at https://papers.ssrn.com/sol3/papers.cfm?abstract_id=4632693

A. Endo, K. Kubota, T. Ito-Sasaki, M. Komatsu, T. Iwama, H. Shiku, K. Y. Inoue, Electrochemistry, 92, 022013 (2024).

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