Dual-Target Immunosensor Based on SERS for Simultaneous Sensing of Hemoglobin and Glycated Hemoglobin in Human Whole Blood

Glycated hemoglobin (HbA1c) refers to the product of the nonenzymatic reaction of hemoglobin (Hb) with glucose and is clinically routine for assessing long-term glycemic control by monitoring its proportion relative to total Hb. However, exclusive reliance on the absolute amount of HbA1c may misjudge patients’ glycemic status; moreover, current commercial detection methods are constrained by the need for specialized equipment/operators or high per-test costs. These two limitations underscore the urgency to develop rapid, portable, and low-cost point-of-care testing (POCT) strategies. Herein, we propose a novel dual-target surface-enhanced Raman scattering (SERS) biosensor based on a magnetic bead-assisted sandwich immunoassay for the simultaneous detection of HbA1c and Hb. Target biomarkers in highly diluted whole blood are specifically captured by antibody-modified magnetic beads, enabling rapid enrichment and complex matrix separation. Subsequently, silica-coated gold nanoparticles (Au@SiO 2 ) embedded with IR-808 and NBA reporters bind to the captured targets, forming a stable sandwich structure. Meanwhile, a 785 nm laser (near-infrared region) was selected to avoid fluorescence interference from the biological matrix and minimize fluorescence-induced spectral overlap, thereby improving detection sensitivity. Notably, the developed method exhibited a favorable linear correlation (R 2 = 0.979) with clinical high-performance liquid chromatography (HPLC) measurements, confirming its reliability. It further enables direct, simultaneous quantitative detection of HbA1c and Hb in human whole blood with minimal sample preprocessing, supporting a more accurate evaluation of HbA1c levels. Furthermore, the method requires <10 μL sample volume and involves a simple one-step mixing procedure, which not only enhances patient comfort but also demonstrates significant potential for future POCT applications.