Choi Jun Hee, Kim Young Hee, Kim Jiwoo, Lim Yong Jun, Kim Min Jung, Bong Ki Wan
Department of Chemical and Biological Engineering, Korea University, Seoul, 02841, South Korea.
Department of Pediatrics, Yongin Severance Hospital, Yonsei University College of Medicine, Yongin-si, 16995, South Korea.
Analyst. 2025 May 12;150(10):2118-2127. doi: 10.1039/d5an00078e.
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. 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 within human serum, which could not be detected using previous methods.
蛋白质作为免疫调节、体内平衡和新陈代谢的介质发挥着关键作用，因此对其进行定量分析对于理解生物医学研究和临床诊断中的疾病机制至关重要。然而，传统方法用于检测临床样本中的蛋白质时，在灵敏度、动态范围和多重检测能力方面存在困难。在本研究中，我们基于编码水凝胶微粒（MPs）并利用酪胺信号放大（TSA）技术开发了一种高灵敏度的多重免疫分析方法。编码水凝胶微粒的大量多重检测能力与酪胺的信号放大作用相结合，实现了高灵敏度的多重免疫分析。通过采用TSA，我们能够获得具有更高特异性的更大检测信号。我们通过封闭捕获抗体偶联步骤后残留的未反应丙烯酸酯双键，有效降低了水凝胶网络中的非特异性结合，并使信噪比提高了3倍。此外，我们优化了主要影响检测灵敏度的三个参数：检测抗体浓度、生物素化酪胺浓度和TSA反应时间。这种方法显著提高了检测灵敏度，检测限低至58 fg/mL。与之前的方法相比，检测灵敏度提高了10倍。此外，通过检测细胞因子IL-4、IL-5、IL-6、IL-9和IL-17验证了该分析方法的多重检测能力，未观察到交叉反应。最后，凭借提高的灵敏度，我们通过在人血清中成功地对低至数百fg/mL浓度的这些细胞因子进行多重检测，证明了我们平台的临床适用性，而使用之前的方法无法检测到这些细胞因子。
