Rationally Engineered Molecularly Imprinted Nanoprobe Enables Facile and Label-Free Targeted Aggregation-Enhanced Raman Assay of HbA1c

Abstract Glycated hemoglobin (HbA1c) is an important clinical indicator for the diagnosis and therapeutic monitoring of diabetes mellitus. However, the existing analytical methods, including liquid chromatography, immunoassay, and mass spectrometry, heavily rely on column resolution, extra labeling bioreagents, or costly instruments, making them incompatible with the needs of point-of-care testing (POCT). Herein, we report a label-free targeted aggregation-enhanced Raman assay for the fast detection of HbA1c. This approach relies on a molecularly imprinted nanoprobe for specific recognition and high-selectivity detection of HbA1c. This nanoprobe is synthesized by rationally selecting a characteristic glycation fragment of HbA1c, which is the key structural difference that distinguishes HbA1c from Hb, as the template and precisely controlling the imprinting layer thickness on a Raman-enhancing core substrate, thereby enabling both selective recognition and near-field Raman signal enhancement. When the nanoprobe interacts with HbA1c, it selectively triggers the aggregation of HbA1c with the nanoprobe. HbA1c molecules are thereby confined in the “hot spot” zones, and their intrinsic Raman signals are enhanced for direct readout without extra labeling. Due to the high sensitivity of enhanced Raman, in the test sample after 104-fold dilution, HbA1c at the ng/mL level is detectable, and its fingerprint information is simultaneously obtained. The method requires less sample consumption, simple operational steps, and rapid readouts. Furthermore, it fulfills the critical need for simultaneous acquisition of quantitative and structural information in a single measurement, offering a powerful analytical tool for in vitro diagnostics and next-generation POCT platforms.