{"title":"通过 LDI-TOF MS 中的质量信号放大,增强抗体功能化金纳米粒子的合成,以实现外泌体的多重检测。","authors":"Gaon Jo, Woon-Seok Yeo","doi":"10.1007/s44211-024-00604-9","DOIUrl":null,"url":null,"abstract":"<div><p>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.</p><h3>Graphical abstract</h3>\n<div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>","PeriodicalId":7802,"journal":{"name":"Analytical Sciences","volume":null,"pages":null},"PeriodicalIF":1.8000,"publicationDate":"2024-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Enhanced synthesis of antibody-functionalized gold nanoparticles for multiplexed exosome detection via mass signal amplification in LDI-TOF MS\",\"authors\":\"Gaon Jo, Woon-Seok Yeo\",\"doi\":\"10.1007/s44211-024-00604-9\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>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. 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引用次数: 0
摘要
我们提出了一种利用有机无基质激光解吸电离飞行时间质谱(LDI-TOF MS)和具有信号放大质量标签(Am-tags)功能的金纳米粒子(AuNPs)灵敏检测外泌体蛋白质的新方法。目标外泌体被AuNPs和生物芯片上的特异性抗体捕获,其中抗体呈现的AuNPs(Ab/Am-tag@AuNPs)含有过量的Am-tags。LDI-TOF质谱分析显示了Ab/Am-tag@AuNPs上Am-tags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-tags 放大,从而提高了灵敏度。我们优化了制备稳定的抗体/氨标记@AuNPs的方案,重点是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共轭的抗体量等参数。随后,我们利用抗 Rab5 固定化金芯片和抗 CD63/Am-tag@AuNPs 以及 LDI-TOF MS 分析,评估了我们的方法检测从 NIH3T3、MCF7 和 HeLa 三种细胞系中分离出来的外泌体的能力。为三种细胞系构建的校准曲线显示出良好的线性关系和检测限。最后,我们强调了使用两种 Am-tags 定量检测外泌体蛋白 CD63 和粘蛋白 1 (MUC1) 的方法的多功能性。LDI-TOF MS 分析表明,CD63 和 MUC1 在 HeLa 和 MCF7 癌细胞中有不同的表达水平。我们的研究结果清楚地表明,Ab/Am-tag@AuNPs 有潜力成为鉴定外泌体中生物标记物的一种灵敏可靠的方法,为生物医学研究和临床应用提供了宝贵的见解。
Enhanced synthesis of antibody-functionalized gold nanoparticles for multiplexed exosome detection via mass signal amplification in LDI-TOF MS
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.
期刊介绍:
Analytical Sciences is an international journal published monthly by The Japan Society for Analytical Chemistry. The journal publishes papers on all aspects of the theory and practice of analytical sciences, including fundamental and applied, inorganic and organic, wet chemical and instrumental methods.
This publication is supported in part by the Grant-in-Aid for Publication of Scientific Research Result of the Japanese Ministry of Education, Culture, Sports, Science and Technology.