| Summary: | Abstract Nowadays, plasmonic biosensors have achieved the limit of detection (LOD) far beyond the clinical or professional standards in the sensing field through material and structural optimizations. However, it remains predominantly a research tool due to the challenge to simultaneously achieve mass production, ultrasensitive and rapid detection, high‐reproducibility, and ease of integration, which are extremely desired in point‐of‐care testing (POCT)‐based commercial products. Here, a label‐free, ultrasensitive, and rapid assay protocol is described for quantitative protein detections in serum samples, which integrates an ultrasmooth gold nanogroove arrays (UGNA) biosensor with an initial rate analysis (IRA) method. i) Compared with the existing plasmonic biosensors, miscellaneous‐protein‐mixed scheme substantially accelerates the binding kinetics of analytes by passivation of all channel inner surfaces. Combined with the IRA method, a wide linear range of AFP detection (1–104 ng mL−1) is obtained, and an enhancement factor of ≈160‐fold in detection time (≈70 s) is achieved for an analyte with ultralow concentration. ii) UGNA can achieve an extremely high surface figure of merit (FOMsurf) under normal incidence, which is immensely useful for miniaturization and multiplexing. iii) UGNA are fabricated using a high‐reproducibility template‐stripping technique, which potentially enables low‐cost mass production. These unique advantages suggest that the biosensor system and analysis method have tremendous potential in POCT biosensing devices.
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