Lee Seungah, Lee Shinae, Ko Young-Ho, Jung Hyungil, Kim Jung Dong, Song Joon Myong, Choo Jaebum, Eo Seong Kug, Kang Seong Ho
Department of Chemistry and Research Institute of Physics and Chemistry (RINPAC), Chonbuk National University, 664-14, 1-Ga, Duckjin-Dong, Duckjin-Gu, Jeonju 561-756, South Korea.
Talanta. 2009 Apr 30;78(2):608-12. doi: 10.1016/j.talanta.2008.12.018. Epub 2008 Dec 24.
We report a method for the quantitative analysis of human serum leptin, which is a protein hormone associated with obesity, using a nanoarray protein chip based on a single-molecule sandwich immunoassay. The nanoarray patterning of a biotin-probe with a spot diameter of 150 nm on a self-assembled monolayer functionalized by MPTMS on a glass substrate was successfully accomplished using atomic force microscopy (AFM)-based dip-pen nanolithography (DPN). Unlabeled leptin protein molecules in human serum were detected based on the sandwich fluorescence immunoassay by total internal reflection fluorescence microscopy (TIRFM). The linear regression equation for leptin in the range of 100 zM-400 aM was determined to be y=456.35 x+80,382 (R=0.9901). The accuracy and sensitivity of the chip assay were clinically validated by comparing the leptin level in adult serum obtained by this method with those measured using the enzyme-linked immunosorbent assay (ELISA) performed with the same leptin standards and serum samples. In contrast to conventional ELISA techniques, the proposed chip methodology exhibited the advantages of ultra-sensitivity, a smaller sample volume and faster analysis time.
我们报告了一种基于单分子夹心免疫分析的纳米阵列蛋白质芯片定量分析人血清瘦素的方法，瘦素是一种与肥胖相关的蛋白质激素。利用基于原子力显微镜（AFM）的蘸笔纳米光刻（DPN）技术，成功地在玻璃基底上由MPTMS功能化的自组装单分子层上制备了直径为150 nm的生物素探针纳米阵列图案。基于全内反射荧光显微镜（TIRFM）的夹心荧光免疫分析检测人血清中未标记的瘦素蛋白分子。确定瘦素在100 zM - 400 aM范围内的线性回归方程为y = 456.35x + 80382（R = 0.9901）。通过将该方法获得的成人血清中瘦素水平与使用相同瘦素标准品和血清样品进行酶联免疫吸附测定（ELISA）测得的水平进行比较，对芯片检测的准确性和灵敏度进行了临床验证。与传统ELISA技术相比，所提出的芯片方法具有超灵敏度、更小样本量和更快分析时间的优点。
