Tyramide signal amplification for a highly sensitive multiplex immunoassay based on encoded hydrogel microparticles

Proteins play a crucial role as mediators of immune regulation, homeostasis, and metabolism, making their quantification essential for understanding disease mechanisms in biomedical research and clinical diagnostics. However, conventional methods when used to detect proteins in clinical samples exhibit difficulty in terms of sensitivity, dynamic range, and multiplex capacity. In this study, we developed a highly sensitive multiplex immunoassay based on encoded hydrogel microparticles (MPs) utilizing tyramide signal amplification (TSA). The combination of the large multiplexing capacity of encoded hydrogel microparticles and the signal amplification of tyramide enables a highly sensitive multiplex immunoassay. By employing TSA, we are able to achieve larger detection signals with higher specificity. We effectively decreased the non-specific binding in the hydrogel network by blocking the unreacted acrylate double bonds remaining after the capture antibody-conjugation step and acquired a 3-fold increase in the signal-to-noise ratio. Also, we optimized three parameters mainly affecting the assay sensitivity: the detection antibody concentration, the biotinyl tyramide concentration, and the TSA reaction time. This approach leads to a significant improvement in assay sensitivity, achieving a limit of detection as low as 58 fg mL^-1. Compared to the previous method, the assay sensitivity is enhanced 10-fold. In addition, the multiplex capability of the assay is validated by detecting cytokines IL-4, IL-5, IL-6, IL-9, and IL-17, with no observed cross-reactivity. Finally, with enhanced sensitivity, we demonstrate the clinical applicability of our platform by successfully multiplexing these cytokines at concentrations down to several hundreds of fg mL^-1 within human serum, which could not be detected using previous methods.