A Rational Framework for Optimizing Electrochemical Biosensor Performance via Systematically Tuning Linker Length and Probe Density on a Nanobody−MXene Platform

Abstract Electrochemical biosensors hold immense promise for sensitive, point-of-care detection of clinically relevant biomarkers from liquid biopsies; however, their performance is fundamentally constrained by charge screening and steric hindrance at the biointerface, particularly under physiological conditions where the Debye length is <1 nm. Herein, we establish a rational framework for engineering high-performance electrochemical biointerfaces by independently tuning two key parameters: linker-based bioreceptor spacing and probe loading density. This strategy is enabled by a modular nanobody−MXene platform, combining 2D MXene nanosheets with covalently conjugated nanobodies. Using anti-GFP nanobodies as model bioreceptors, we identified an intermediate linker regime that maximized signal transduction and a low-density probe regime that mitigated steric hindrance while enhancing dynamic range and sensitivity. Applied to a VHH72 nanobody−MXene biosensor, this framework enabled the detection of SARS-CoV-2 S1 with a 12 pM limit of detection, high specificity, and robust performance in saliva. Collectively, this work establishes a dual-parameter framework for advancing next-generation electrochemical biosensors.