Development of Insulin Sensors Based on Faradaic and Non-Faradaic Electrochemical Impedance Spectroscopy Detection Principle Using Single Chain Antibody (scFv)

Introduction In this paper, we report novel insulin sensors using insulin single-chain antibody (scFv) based on two electrochemical impedance spectroscopy (EIS) methods; faradaic EIS and non-faradaic EIS. For faradaic EIS measurement, varieties of molecular recognition elements (MREs) can be utilized, such as IgG and binding proteins, without consideration of their molecular size. However, a solubilized redox probe is necessary to aid the transfer of electrons and detection is based on the flux of electrons and the obstruction that is present on the electrode surface. Non-faradaic EIS does not require a solubilized redox probe and is based around changes within the electrical double layer. However, there are the limitation of the size for MREs, since the ideal size of MREs should be smaller than the size of electrical double layer (EDL). In this paper, thanks to the small size of scFv as MRE for EIS, we construct two different types of EIS based insulin sensors, and compare the suitability in the use for insulin monitoring, being dedicated for the management of diabetic patients. Methods Development of this sensor requires immobilization of a molecular recognition element (MRE) that binds with insulin. For this purpose, we have cultivated single-chain insulin antibody by transforming an Escherichia coli strain, harvesting and filtering the protein culture, and purifying the protein culture. Through this cultivation procedure, we obtained insulin scFv that can be immobilized on a gold surface electrode. For faradaic EIS measurement, we employed potassium ferricyanide as the solubilized redox probe to conduct insulin measurement. Binding of insulin concentrations with insulin scFv caused a change in electron flux, which thereby caused changes in charge-transfer resistance (Rct) or impedance. In non-faradaic EIS measurement, ionic liquid is typically used as the buffer to redistribute the dielectric bilayer. Thanks to the small size of insulin scFv, capacitance changes can be directly measured within EDL. Both detection principles were compared by selecting an optimized frequency which best reflects binding between insulin and the MRE. Results We were able to identify an optimized frequency for both detection principles when measuring insulin, establishing a unique change in impedance. A change in Rct was noticed through faradaic EIS when measuring insulin against the insulin scFv. Additionally, at the optimized frequency the sensor had a slope of 701 Ohms/Ln(nM). With the non-Faradaic EIS detection principle, the sensor displayed changes within EDL. Discussion We were able to determine that faradaic and non-faradaic EIS could be used to measure changing insulin concentrations when using insulin scFv as the MRE. Differences between both detection principles existed due to the use of a solubilized redox probe for faradaic EIS versus the absence of it when conducing non-Faradaic EIS. Future work will include comparing the sensitivity and selectivity of both detection methods when measuring insulin. References [1] A. Malkoc et al., “Enhancing Glycemic Control via Detection of Insulin Using Electrochemical Impedance Spectroscopy,” J. Diabetes Sci. Technol., vol. 11, no. 5, pp. 930–935, Mar. 2017, doi: 10.1177/1932296817699639. [2] T. L. Adamson, F. A. Eusebio, C. B. Cook, and J. T. LaBelle, “The promise of electrochemical impedance spectroscopy as novel technology for the management of patients with diabetes mellitus,” The Analyst, vol. 137, no. 18, pp. 4179–4187, Sep. 2012, doi: 10.1039/c2an35645g. [3] L. P. Malpiedi, C. A. Díaz, B. B. Nerli, and A. Pessoa, “Single-chain antibody fragments: Purification methodologies,” Process Biochem., vol. 48, no. 8, pp. 1242–1251, Aug. 2013, doi: 10.1016/j.procbio.2013.06.008. [4] A. S. Tanak, B. Jagannath, Y. Tamrakar, S. Muthukumar, and S. Prasad, “Non-faradaic electrochemical impedimetric profiling of procalcitonin and C-reactive protein as a dual marker biosensor for early sepsis detection,” Anal. Chim. Acta X, vol. 3, p. 100029, Nov. 2019, doi: 10.1016/j.acax.2019.100029.