(Invited) Development of Electrochemical 6-Well Plate for Immunosensors

Abstract

While developing low-cost and multiplexing electrochemical (EC) devices for bioassay is very attractive, the miniaturization of reference electrodes (RE) is a critical issue for fabricating EC devices. Herein, a polymer-based EC device, named EC 6-well plate, is proposed and fabricated using a non-photolithography method. Polyethylene terephthalate glycol (PETG) is used as a substrate and laser-cut polyester (PET) film is used as a mask for patterning the electrodes. Acrylic mold with wells (60 μL) was bonded to the PETG substrate. Schematic diagrams (Figure 1) are used to illustrate the preparation of electrochemical 6-well plates. The diameter of the working electrode (WE) is 900 μm, and each WE-modifying step only requires 1 μL of reagent.Both electrochemical (HCl method) and chemical methods (FeCl3 method) were proposed to prepare the solid-state Ag/AgCl RE. Precise time control is critical for the preparation procedure. It is hard to control the time in 1 s or under 1 s using the chemical method. Hence, the electrochemical method was selected for continued work since the reaction time can be precisely controlled. The stability of solid-state Ag/AgCl and drift of open circuit potential (OCP) can be affected by anions present in the background electrolyte. Herein, 0.1 M KCl, 1 M KCl, and 0.1 M KNO3 containing 5 mM K3[Fe(CN)6]/K4 [Fe(CN)6] were compared as the electrolytes. Considering that 0.01 M PBS is the most commonly used buffer solution for bioassay, 0.01 M PBS containing 5 mM K3[Fe(CN)6]/K4 [Fe(CN)6] was also included. OCP was recorded in these electrolytes for 1 hour. These results revealed that 0.1 M KCl is the best choice for the background electrolyte. The solid-state Ag/AgCl RE-based three-electrode system, the all Au three-electrode system (3E), and all Au two-electrode system (2E) were used to develop the immunosensor forClostridium difficile toxin B detection. Differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) were applied to test the stability of the EC immunosensor. The three Au electrodes system showed just a minor DPV peak position shift and the standard deviation (SD) of OCP is about 0.0026 mV. The two Au electrodes system showed no DPV peak position shift and the SD of OCP is 0.32 mV. The solid-state Ag/AgCl RE-based three-electrode system showed a relatively large DPV peak position shift and the SD of OCP is about 4.6 mV. It is demonstrated that the all Au three-electrode system is superior to the solid-state Ag/AgCl RE-based three-electrode system for developing an immunosensor. Figure 1