Four Assay Designs and On-Chip Calibration: Gadgets for a Sepsis Protein Array
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- 作者：Patricia Buchegger ; Claudia Preininger
- 刊名：Analytical Chemistry
- 出版年：2014
- 出版时间：March 18, 2014
- 年：2014
- 卷：86
- 期：6
- 页码：3174-3180
- 全文大小：307K
- 年卷期：v.86,no.6(March 18, 2014)
- ISSN：1520-6882
文摘
A protein microarray for the early stage diagnosis of sepsis that allows the simultaneous detection of C-reactive protein (CRP) (2鈥?00 渭g/mL), procalcitonin (PCT) (0.2鈥?0 ng/mL), and interleukin 6 (IL-6) (2鈥?000 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 鈥渓ow-sensitive鈥?sandwich assay for CRP. The calibration is integrated directly on the chip resulting in a 鈥渙ne patient鈥搊ne array鈥?format, to provide a user-friendly and rapid diagnostic tool. Four different assay designs are introduced: (I) the classical assay that works with biotin鈥搒treptavidin chemistry, (II) the rapid assay that is performed in a single detection step, and two ultrasensitive assay designs accomplished either by (III) 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鈥?4%; CVinter: 18鈥?4%), while the sensitivity (limits of detection (LODs)) increased by 1 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.