Protein binding assessment of immobilized nanobody using interferometric nanoporous platform

Abstract Protein sensors are key tools for infectious disease diagnosis and monitoring. The medical diagnostics field is now rapidly identifying key protein markers for the detection of a range of conditions, from early cancer development through to assessment of dementia risk a decade prior to symptoms onset. Many point‐of‐use diagnostic tools form part of standard protocols for small molecule sensing in healthcare, including glucose and ketone body sensors. Label‐free optical biosensors have emerged as reliable detection tools that provide high versatility and adaptability to detect a broad range of target analytes. Herein, we combined nanoporous anodic alumina (NAA) photonic chip technology with tailor‐engineered “nanobodies”—recombinant variable domains of heavy‐chain‐only antibodies—to achieve high affinity binding to human serum albumin (HSA). We monitored dynamic shifts in the optical fingerprint of nanobody‐conjugated NAA platforms in real time when these were exposed to target proteins, through reflectometric interference spectroscopy (RIfS). We performed a comprehensive characterization of the sensing performance, where binding mechanisms were elucidated through kinetic profiles. The nanobody‐conjugated NAA protein sensor demonstrated good affinity, selectivity, and sensitivity toward HSA. Our analysis revealed a sensitivity of 27.3 ± 3.2 nm µM −1 and low limit of detection of 16.3 ± 1.6 µM, which are well‐below the ranges for diagnosis of medical conditions such as nephrotic syndrome. The obtained results revealed that the RIfS‐based protein sensor integrated with nanobody‐modified NAA has promising potential to develop point‐of‐care platforms for clinical diagnosis of diseases.