Abstract：
Multiplex detection of low-abundance protein biomarkers in biofluids can contribute to diverse biomedical fields such as early diagnosis and precision medicine. However, conventional techniques such as digital ELISA, microarray, and hydrogel-based assay still face limitations in terms of efficient protein detection due to issues with multiplexing capability, sensitivity, or complicated assay procedures. In this study, we present the degassed micromold-based particle isolation technique for highly sensitive and multiplex immunoassay with enzymatic signal amplification. Using degassing treatment of nanoporous polydimethylsiloxane (PDMS) micromold, the encoded particles are isolated in the mold within 5 min absorbing trapped air bubbles into the mold by air suction capability. Through 10 min of signal amplification in the isolated spaces by fluorogenic substrate and horseradish peroxidase labeled in the particle, the assay signal is amplified with one order of magnitude compared to that of the standard hydrogel-based assay. Using the signal amplification assay, vascular endothelial growth factor (VEGF) and chorionic gonadotropin beta (CG beta), the preeclampsia-related protein biomarkers, are quantitatively detected with a limit of detection (LoD) of 249 fg/mL and 476 fg/mL in phosphate buffer saline. The multiplex immunoassay is conducted to validate negligible non-specific detection signals and robust recovery rates in the multiplex assay. Finally, the VEGF and CG beta in real urine samples are simultaneously and quantitatively detected by the developed assay. Given the high sensitivity, multiplexing capability, and process simplicity, the presented particle isolation-based signal amplification assay holds significant potential in biomedical and proteomic fields.
摘要：
生物流体中低丰度蛋白质生物标志物的多重检测可以为早期诊断和精准医学等多种生物医学领域做出贡献。然而,传统技术，如数字ELISA，微阵列,和基于水凝胶的测定在有效的蛋白质检测方面仍然面临限制，由于多路复用能力的问题，灵敏度,或复杂的化验程序。在这项研究中,我们提出了基于脱气微模具的颗粒分离技术，用于具有酶信号放大的高灵敏度和多重免疫测定。对纳米多孔聚二甲基硅氧烷(PDMS)微模具进行脱气处理,编码的颗粒在5分钟内被隔离在模具中,通过空气抽吸能力将捕获的气泡吸收到模具中。通过荧光底物和颗粒中标记的辣根过氧化物酶在隔离空间中进行10分钟的信号放大，与标准的基于水凝胶的测定相比,测定信号被放大一个数量级。使用信号放大分析，血管内皮生长因子（VEGF）和绒毛膜促性腺激素β（CGβ），先兆子痫相关的蛋白质生物标志物，在磷酸盐缓冲盐水中以249fg/mL和476fg/mL的检测限(LoD)进行定量检测。进行多重免疫测定以验证多重测定中可忽略的非特异性检测信号和稳健的回收率。最后,通过开发的测定法同时定量检测真实尿液样品中的VEGF和CGβ。由于灵敏度高,复用能力，和过程简单，提出的基于粒子分离的信号放大试验在生物医学和蛋白质组学领域具有重要的潜力。
