{"title":"通过共靶向四泛素生物标记物加强细胞外囊泡检测","authors":"Jesus M Lopez Baltazar, Wenchao Gu, Qiuming Yu","doi":"10.1021/acs.analchem.4c04086","DOIUrl":null,"url":null,"abstract":"<p><p>Extracellular vesicles (EVs) are emerging as key diagnostic biomarkers due to their widespread presence in body fluids and the proteins on their surfaces, which reflect the identity and condition of their parent cells. Research has focused on detecting EVs with biosensors that target individual transmembrane proteins (TMPs) like tetraspanins. However, due to TMP heterogeneity and the formation of tetraspanin-enriched microdomains (TEMs), cotargeting multiple TMPs is a promising strategy for enhancing EV detection. In this work, we introduce a dual-antibody surface functionalization approach using surface plasmon resonance (SPR) biosensors to cotarget tetraspanins on EVs derived from mouse macrophages. The expression of EV tetraspanin markers followed the trend of CD9 > CD63 > CD81, which was consistent with the EV detection targeting their nontetraspanin partners, exhibiting LFA-1 > ICAM-1 > VCAM-1, and suggesting a differential role of tetraspanins with their associated TMPs. Cotargeting EV tetraspanins <i>via</i> CD81/CD63, CD81/CD9, and CD63/CD9 dual monoclonal antibody surfaces resulted in higher EV detection compared to predictions based on binding with two monoclonal antibodies against tetraspanins without cotargeting. Furthermore, the optimization of dual monoclonal antibody surface ratios to improve cotargeting effect yielded a statistically significant enhancement in the sensitivity of EV detection. These findings underscore the importance of TEMs in designing EV-based biosensing platforms to achieve optimized sensitivity in EV detection.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7000,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Enhancing Extracellular Vesicle Detection <i>via</i> Cotargeting Tetraspanin Biomarkers.\",\"authors\":\"Jesus M Lopez Baltazar, Wenchao Gu, Qiuming Yu\",\"doi\":\"10.1021/acs.analchem.4c04086\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Extracellular vesicles (EVs) are emerging as key diagnostic biomarkers due to their widespread presence in body fluids and the proteins on their surfaces, which reflect the identity and condition of their parent cells. Research has focused on detecting EVs with biosensors that target individual transmembrane proteins (TMPs) like tetraspanins. However, due to TMP heterogeneity and the formation of tetraspanin-enriched microdomains (TEMs), cotargeting multiple TMPs is a promising strategy for enhancing EV detection. In this work, we introduce a dual-antibody surface functionalization approach using surface plasmon resonance (SPR) biosensors to cotarget tetraspanins on EVs derived from mouse macrophages. The expression of EV tetraspanin markers followed the trend of CD9 > CD63 > CD81, which was consistent with the EV detection targeting their nontetraspanin partners, exhibiting LFA-1 > ICAM-1 > VCAM-1, and suggesting a differential role of tetraspanins with their associated TMPs. Cotargeting EV tetraspanins <i>via</i> CD81/CD63, CD81/CD9, and CD63/CD9 dual monoclonal antibody surfaces resulted in higher EV detection compared to predictions based on binding with two monoclonal antibodies against tetraspanins without cotargeting. Furthermore, the optimization of dual monoclonal antibody surface ratios to improve cotargeting effect yielded a statistically significant enhancement in the sensitivity of EV detection. These findings underscore the importance of TEMs in designing EV-based biosensing platforms to achieve optimized sensitivity in EV detection.</p>\",\"PeriodicalId\":27,\"journal\":{\"name\":\"Analytical Chemistry\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":6.7000,\"publicationDate\":\"2024-10-03\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Analytical Chemistry\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1021/acs.analchem.4c04086\",\"RegionNum\":1,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"CHEMISTRY, ANALYTICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical Chemistry","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1021/acs.analchem.4c04086","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
引用次数: 0
摘要
由于细胞外囊泡(EVs)广泛存在于体液中,而且其表面的蛋白质能反映母细胞的特征和状况,因此正在成为关键的诊断生物标记物。研究的重点是利用针对单个跨膜蛋白(TMP)(如四跨蛋白)的生物传感器来检测 EV。然而,由于 TMP 的异质性和四跨蛋白富集微域(TEM)的形成,共靶向多个 TMP 是增强 EV 检测的一种有前途的策略。在这项工作中,我们利用表面等离子体共振(SPR)生物传感器引入了一种双抗体表面功能化方法,以共同靶向小鼠巨噬细胞EV上的四跨蛋白。EV四跨蛋白标记物的表达遵循CD9 > CD63 > CD81的趋势,这与针对其非四跨蛋白伙伴的EV检测结果一致,表现为LFA-1 > ICAM-1 > VCAM-1,表明四跨蛋白与其相关的TMPs具有不同的作用。通过 CD81/CD63、CD81/CD9 和 CD63/CD9 双单克隆抗体表面共同靶向 EV 四跨蛋白,与不共同靶向的情况下根据两种针对四跨蛋白的单克隆抗体结合预测的结果相比,EV 的检测率更高。此外,优化双单克隆抗体表面比例以提高共靶效应,在统计学上显著提高了检测 EV 的灵敏度。这些发现强调了 TEM 在设计基于 EV 的生物传感平台以优化 EV 检测灵敏度方面的重要性。
Enhancing Extracellular Vesicle Detection via Cotargeting Tetraspanin Biomarkers.
Extracellular vesicles (EVs) are emerging as key diagnostic biomarkers due to their widespread presence in body fluids and the proteins on their surfaces, which reflect the identity and condition of their parent cells. Research has focused on detecting EVs with biosensors that target individual transmembrane proteins (TMPs) like tetraspanins. However, due to TMP heterogeneity and the formation of tetraspanin-enriched microdomains (TEMs), cotargeting multiple TMPs is a promising strategy for enhancing EV detection. In this work, we introduce a dual-antibody surface functionalization approach using surface plasmon resonance (SPR) biosensors to cotarget tetraspanins on EVs derived from mouse macrophages. The expression of EV tetraspanin markers followed the trend of CD9 > CD63 > CD81, which was consistent with the EV detection targeting their nontetraspanin partners, exhibiting LFA-1 > ICAM-1 > VCAM-1, and suggesting a differential role of tetraspanins with their associated TMPs. Cotargeting EV tetraspanins via CD81/CD63, CD81/CD9, and CD63/CD9 dual monoclonal antibody surfaces resulted in higher EV detection compared to predictions based on binding with two monoclonal antibodies against tetraspanins without cotargeting. Furthermore, the optimization of dual monoclonal antibody surface ratios to improve cotargeting effect yielded a statistically significant enhancement in the sensitivity of EV detection. These findings underscore the importance of TEMs in designing EV-based biosensing platforms to achieve optimized sensitivity in EV detection.
期刊介绍:
Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.