Four Assay Designs and On-Chip Calibration: Gadgets for a Sepsis Protein Array
Four Assay Designs and On-Chip Calibration: Gadgets for a Sepsis Protein Array
复制标题
DOI：
10.1021/ac5000784
复制
发表时间：
2014-03-18
影响因子：
7.4
通讯作者：
Preininger, Claudia
中科院分区：
文献类型：
--
作者：
Buchegger, Patricia;Preininger, Claudia
一种用于脓毒症早期诊断的蛋白质芯片可以同时检测c反应蛋白（CRP） （2-200 μ g/mL）、降钙素原（PCT） （0.2-50 ng/mL）和白细胞介素6 (IL-6) （2-2000 pg/mL）。为了能够平行检测不同丰度的分析物，CRP和磷胆碱之间的低结合亲和力被利用在CRP的“低敏感”夹心法中。校准直接集成在芯片上，形成“一病人一阵列”格式，提供用户友好的快速诊断工具。介绍了四种不同的分析设计：(I)与生物素链亲和素化学一起工作的经典分析，（II）在单个检测步骤中执行的快速分析，以及通过（111）酶或（IV）抗体介导扩增导致高密度标记的两种超灵敏分析设计。通过反复测量市售的经认证的对照血清的低、中、高浓度水平来评估分析设计。所有试验设计的精密度相似(变异系数（CV）， CVintra 8-14%；CVinter: 18-34%)，而超灵敏试验（III， IV）的灵敏度（检出限（lod））提高了1个数量级，准确性与分析物相关，但经典(I)和抗体扩增（IV）试验的准确性最好。
A protein microarray for the early stage diagnosis of sepsis that allows the simultaneous detection of C-reactive protein (CRP) (2-200 mu g/mL), procalcitonin (PCT) (0.2-50 ng/mL), and interleukin 6 (IL-6) (2-2000 pg/mL) has been developed. To enable the parallel detection of the differently abundant analytes, the low binding affinity between CRP and phosphocholine is exploited in a "low-sensitive" sandwich assay for CRP. The calibration is integrated directly on the chip resulting in a "one patient one array" format, to provide a user-friendly and rapid diagnostic tool. Four different assay designs are introduced: (I) the classical assay that works with biotin streptavidin chemistry, (II) the rapid assay that is performed in a single detection step, and two ultrasensitive assay designs accomplished either by (111) an enzymatic or (IV) an antibody mediated amplification resulting in high density labeling. The assay designs were evaluated by the repetitive measurement of low, medium, and high concentration levels of commercially available certified control sera. The precision was similar across all assay designs (coefficient of variation (CV), CVintra 8-14%; CVinter : 18-34%), while the sensitivity (limits of detection (LODs)) increased by I order of magnitude for the ultrasensitive assays (III, IV) and the accuracy was analyte dependent but best for the classical (I) and the antibody amplified (IV) assays.