Tyramide signal amplification integrated with magnetic bead separation to overcome the matrix effect and detect SAA protein in peripheral blood

Abstract

Complex clinical serum samples often induce severe matrix effects during immunoassay quantification. While substantial sample dilution is a practical solution to mitigate this interference, it significantly reduces the concentration of target analytes. This reduction makes already low-abundance proteins highly difficult to detect using traditional methods. To address this challenge, this study integrated carboxyl magnetic bead enrichment with the tyramide signal amplification (TSA) platform to enable high-fold dilution. In this system, the magnetic beads capture and isolate plasma biomarker proteins while the enzymatic cascade amplifies the detection signal, thereby mitigating matrix interference while quantifying trace plasma biomarker proteins. We utilized serum amyloid A (SAA) as a model biomarker to verify the analytical reliability of this combined method. In comparison to the classical assay without TSA, which demonstrated a linear range of 3.2 pM–5.0 nM. TSA amplification shifted the linear quantitative range to 74 fM–30 pM, achieving an improved limit of detection (LOD) from 1.1 pM to 25 fM. This sensitivity enables implementation of a high-dilution strategy intended to mitigate matrix interference. Finally, the optimized platform was applied to analyze 66 clinical serum samples. The detection results revealed a distinct upregulation in average maternal SAA levels within the preterm birth (PTB) group compared to the healthy full-term birth (FTB) group. These findings support the reliability of the constructed analytical method and provide preliminary evidence that elevated SAA levels may be associated with PTB.