Liang Ke-Zhong, Qi Jun-Sheng, Mu Wei-Jun, Liu Zheng-Xue
Department of Chemistry and Environment, Chongqing Three Gorges University, 400400, Chongqing, People's Republic of China.
Bioprocess Biosyst Eng. 2009 Apr;32(3):353-9. doi: 10.1007/s00449-008-0254-0. Epub 2008 Aug 2.
Various sensor-based immunoassay methods have been extensively developed for the detection of interleukin-6 (IL6), but most often exhibit low detection signals and low detection sensitivity, and are unsuitable for routine use. The aim of this work is to develop a simple and sensitive conductometric immunoassay for IL6 in human serum by using an organic/inorganic hybrid membrane-functionalized interface. Initially, thionine-bound 3,4,9,10-perylenetetracarboxylic acid was doped into colloidal alumina, then nanogold particles were immobilized onto the thionine surface, and then horseradish peroxidase-labeled anti-IL6 antibodies were conjugated on the nanogold surface. The organic/inorganic hybrid membrane provides a good microenvironment for the immobilization of biomolecules, enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The performance and factors influencing the performance of the immunosensor were evaluated. The detection is based on the change in local conductivity before and after the antigen-antibody interaction in 0.02 M phosphate buffer solution (pH 6.8) containing 50 microM H(2)O(2), 0.01 M KI and 0.15 M NaC1. Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 25 to 400 pg/ml towards IL6 with a relatively low detection limit of 5 pg/ml (S/N = 3). The stability, reproducibility and precision of the immunosensor were acceptable. 37 serum specimens were assayed by the developed immunosensor and standard enzyme-linked immunosorbent assay, respectively, and the results obtained were almost consistent. More importantly, the detection methodology provides a promising approach for other proteins or biosecurity.
为了检测白细胞介素-6（IL6），人们已经广泛开发了各种基于传感器的免疫分析方法，但这些方法大多检测信号低、检测灵敏度低，不适用于常规检测。本研究旨在利用有机/无机杂化膜功能化界面，开发一种简单、灵敏的用于检测人血清中IL6的电导免疫分析方法。首先，将硫堇结合的3,4,9,10-苝四羧酸掺杂到胶体氧化铝中，然后将纳米金颗粒固定在硫堇表面，接着将辣根过氧化物酶标记的抗IL6抗体偶联在纳米金表面。有机/无机杂化膜为生物分子的固定提供了良好的微环境，增加了蛋白质的表面覆盖率，提高了免疫传感器的灵敏度。对免疫传感器的性能及影响其性能的因素进行了评估。检测基于在含有50 microM H(2)O(2)、0.01 M KI和0.15 M NaCl的0.02 M磷酸盐缓冲溶液（pH 6.8）中抗原-抗体相互作用前后局部电导率的变化。在最佳条件下，所提出的免疫传感器对IL6的线性范围为25至400 pg/ml，检测限相对较低，为5 pg/ml（S/N = 3）。免疫传感器的稳定性、重现性和精密度均可接受。分别用所开发的免疫传感器和标准酶联免疫吸附测定法对37份血清标本进行检测，所得结果几乎一致。更重要的是，该检测方法为其他蛋白质或生物安全检测提供了一种有前景的方法。
