A Dual‐Modal Single‐Antibody Plasmonic Spectro‐Immunoassay for Detection of Small Molecules

Abstract Small molecules play a pivotal role in regulating physiological processes and serve as biomarkers to uncover pathological conditions and the effects of therapeutic treatments. However, it remains a significant challenge to detect small molecules given the size as compared to macromolecules. Recently, the newly emerging plasmonic immunoassays based on surface‐enhanced Raman scattering (SERS) offer great promise to deliver extraordinary sensitivity. Nevertheless, they are limited by the intrinsic SERS intensity fluctuations associated with the SERS uncertainty principle. The single transducer that relies on the intensity change is also prone to false signals. Additionally, the prevailing sandwich immunoassay format proves less effective towards detecting small molecules. To circumvent these critical issues, a dual‐modal single‐antibody approach that synergizes both the intensity and shift of the peak‐based immunoassay with Raman enhancement, coined as the INSPIRE assay, is developed for small molecules detection. With two independent transduction mechanisms, it allows better prediction of analyte concentration and attenuation of signal artifacts, providing a new and robust strategy for molecular analysis. With a proof‐of‐concept demonstration for detection of free T4 and testosterone in serum matrix, the authors envision that the INSPIRE assay could be expanded for a wide spectrum of applications in biomedical diagnosis, discovery of new biopharmaceuticals, food safety, and environmental monitoring.