Despite significant progress in the field of biosensing, the impact of electric field on biosensor detection capability and the feasibility of the biosensor application in wastewater has yet to be investigated. The objective of this study was to develop a low-cost, highly sensitive, and selective reduced graphene oxide (rGO)-based biosensor. The constructed biosensor consists of an in-house prepared GO and a four-terminal Kelvin sensing. Spin-coating was chosen as the deposition technique and results revealed an optimal GO number of layers and concentration of 7 and 2 mg/mL, respectively. Experiments to determine the effects of electric field on the performance of the biosensor showed significant changes in the biosensor surface, also presenting a direct impact on the biosensor functionality, such that the biosensor showed an increase in limit of detection (LOD) from 1 to 106 fg/mL when the applied voltage was increased from 0.0008 to 0.2 V. Furthermore, this study successfully explores a pilot scale setup, mimicking wastewater flow through sewage pipelines. The demonstrated improvements in the detection capability and sensitivity of this biosensor at optimized testing conditions make it a promising candidate for further development and deployment for the detection of protein analytes present at very low concentrations in aqueous solutions. In addition, the application of this biosensor could be extended to the detection of protein analytes of interest (such as the spike protein of SARS-CoV-2) in much more complex solutions, like wastewater.
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