我们提出了一种使用无有机基质激光解吸/电离飞行时间质谱 (LDI-TOF MS) 和带有信号放大质量标签 (Am-tags) 功能的金纳米颗粒 (AuNP) 进行灵敏外泌体蛋白检测的新方法。目标外泌体被 AuNP 和生物芯片上的特异性抗体捕获，其中抗体呈递的 AuNP (Ab/Am-tag@AuNPs) 含有过量的 Am 标签。 LDI-TOF MS 分析揭示了 Ab/Am-tag@AuNPs 上 Am-标签的质量信号，表明目标外泌体的存在。因此，目标信号被大量 Am 标签放大，从而提高了灵敏度。我们优化了制备稳定 Ab/Am-tag@AuNPs 的方案，重点关注用于 AuNP 功能化的硫醇分子的浓度和比例、偶联反应的合适溶剂以及与 AuNPs 缀合的抗体量等参数。随后，我们使用抗 Rab5 固定金芯片和抗 CD63/Am-tag@AuNP 并进行 LDI-TOF MS 分析，评估了我们的方法检测从 NIH3T3、MCF7 和 HeLa 三种细胞系分离的外泌体的能力。为三种细胞系构建的校准曲线显示出线性关系和良好的检测限。最后，我们强调了我们的方法使用两种类型的 Am 标签定量检测外泌体蛋白 CD63 和粘蛋白 1 (MUC1) 的多功能性。 LDI-TOF MS 分析显示 HeLa 和 MCF7 癌细胞中存在不同表达水平的 CD63 和 MUC1。我们的研究结果清楚地表明 Ab/Am-tag@AuNPs 作为识别外泌体中生物标志物的敏感而可靠的方法的潜力，为它们在生物医学研究和临床环境中的效用提供了宝贵的见解。© 2024。作者，独家日本分析化学学会许可。

We present a novel method for sensitive exosomal protein detection using organic matrix-free laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) and gold nanoparticles (AuNPs) functionalized with mass tags for signal amplification (Am-tags). Target exosomes were captured by specific antibodies on AuNPs and a biochip, where the antibody-presenting AuNPs (Ab/Am-tag@AuNPs) contained excess Am-tags. LDI-TOF MS analysis revealed the mass signal of Am-tags on Ab/Am-tag@AuNPs, indicating the presence of target exosomes. Thus, the target signal was amplified by a large number of Am-tags, resulting in enhanced sensitivity. We optimized the protocol to prepare stable Ab/Am-tag@AuNPs, focusing on parameters such as the concentration and ratio of thiol molecules for AuNP functionalization, suitable solvents for the coupling reaction, and amount of antibodies conjugated to the AuNPs. Subsequently, we evaluated the ability of our method to detect exosomes isolated from three cell lines, NIH3T3, MCF7, and HeLa, using an anti-Rab5 immobilized gold chip and anti-CD63/Am-tag@AuNPs with LDI-TOF MS analysis. Calibration curves constructed for the three cell lines showed a linear relationship with an excellent limit of detection. Finally, we emphasized the versatility of our method for the quantitative detection of exosomal proteins CD63 and mucin 1 (MUC1) using two types of Am-tags. LDI-TOF MS analysis revealed the presence of CD63 and MUC1 at different expression levels in HeLa and MCF7 cancer cells. Our findings clearly indicate the potential of Ab/Am-tag@AuNPs as a sensitive and reliable approach for identifying biomarkers in exosomes, providing valuable insights into their utility in biomedical research and clinical settings.© 2024. The Author(s), under exclusive licence to The Japan Society for Analytical Chemistry.