Multiplexed femtomolar quantitation of human cytokines in a fluoropolymer microcapillary film
Multiplexed femtomolar quantitation of human cytokines in a fluoropolymer microcapillary film
复制标题
DOI：
10.1039/c5an00238a
复制
发表时间：
2015-01-01
期刊：
影响因子：
4.2
通讯作者：
Reis, Nuno M.
中科院分区：
文献类型：
--
作者：
Castanheira, Ana P.;Barbosa, Ana I.;Reis, Nuno M.
多种细胞因子的灵敏定量可以在感染、炎症和免疫病理学期间提供重要的诊断信息。在这项研究中，使用新型小型化ELISA平台，以单重和多重形式显示了人细胞因子IL-1 β、IL-6、IL-12 p70和TNF α的灵敏免疫测定检测。该平台使用一次性塑料多注射器抽吸器（MSA），其集成8个一次性含氟聚合物微流体测试条，每个测试条包含10个200 μ m平均i.d.用一组单克隆抗体包被的微毛细管。因此，每个MSA设备对8个样品进行10次测试，提供80次测量。使用新型含氟聚合物微流体材料和平板扫描仪中的简单比色检测获得了前所未有的灵敏度水平。单重检测的检测限范围为2.0至15.0 pg ml（-1），即单重细胞因子检测的检测限为35和713 femtomolar，并且测定内和测定间的变异系数（CV）保持在10%以内。此外，还开发了一种三重免疫测定法，用于同时测量给定样本中的IL-1 β、IL-12 p70和TNF α，其浓度范围为pg/ml。这些测定允许高灵敏度测量，快速< 15分钟测定或从未稀释的血清中检测。该系统的便携性、速度和低成本非常适合即时检测和现场诊断应用。
Sensitive quantitation of multiple cytokines can provide important diagnostic information during infection, inflammation and immunopathology. In this study sensitive immunoassay detection of human cytokines IL-1 beta, IL-6, IL-12p70 and TNF alpha is shown for singleplex and multiplex formats using a novel miniaturized ELISA platform. The platform uses a disposable plastic multi-syringe aspirator (MSA) integrating 8 disposable fluoropolymer microfluidic test strips, each containing an array of ten 200 mu m mean i.d. microcapillaries coated with a set of monoclonal antibodies. Each MSA device thus performs 10 tests on 8 samples, delivering 80 measurements. Unprecedented levels of sensitivity were obtained with the novel fluoropolymer microfluidic material and simple colorimetric detection in a flatbed scanner. The limit of detection for singleplex detection ranged from 2.0 to 15.0 pg ml(-1), i.e. 35 and 713 femtomolar for singleplex cytokine detection, and the intra-and inter-assay coefficient of variation (CV) remained within 10%. In addition, a triplex immunoassay was developed for measuring IL-1 beta, IL-12p70 and TNF alpha simultaneously from a given sample in the pg ml(-1) range. These assays permit high sensitivity measurement with rapid < 15 min assay or detection from undiluted blood serum. The portability, speed and low-cost of this system are highly suited to point-of-care testing and field diagnostics applications.