Biomarker Research and Development Centre, Level 5 East, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, QLD 4072, Australia.
Langmuir. 2009 Dec 1;25(23):13510-5. doi: 10.1021/la903148n.
A 10-fold improvement in the signal-to-noise (S/N) ratio of an optically encoded silica particle-based immunoassay was achieved through incorporating a protein resistant poly(ethylene glycol) (PEG) surface layer and optimizing antibody immobilization conditions. PEG was activated using 2,2,2-trifluoroethanesulfonyl chloride (tresyl) and required a minimum reaction time of 1.5 h. The activated PEG had a reactive half-life of approximately 5 h when stored in acidified dimethyl sulfoxide (DMSO). By increasing the protein incubation time and concentration, a maximum antibody loading on the particle surface of 1.6 x 10(-2) molecules per nm(2) was achieved. The assay S/N ratio was assessed using a multiplexed multicomponent optically encoded species-specific immunoassay. Encoded particles were covalently grafted or nonspecifically coated with either bovine or mouse IgG for the simultaneous detection of complementary anti-IgG "target" or uncomplementary anti-IgG "noise". The versatility and potential as a serum-based assay platform was demonstrated by immobilizing either a polyclonal antibody or an engineered single-chain variable fragment (scFv) capture probe on particles for the detection of the ovarian cancer biomarker, mesothelin (MSLN). The MLSN antigen was spiked into PBS buffer or 50% human serum. Both capture probe orientations, and media conditions showed similar low level detection limits of 5 ng/mL; however, a 40% decrease in maximum signal intensity was observed for assays run in 50% serum.
通过在基于二氧化硅颗粒的免疫分析中加入抗蛋白质的聚乙二醇（PEG）表面层并优化抗体固定化条件，可将信号与噪声（S/N）比提高 10 倍。使用 2,2,2-三氟乙磺酰氯（三氟甲磺酸酯）激活 PEG，反应时间至少需要 1.5 小时。当储存在酸化的二甲基亚砜（DMSO）中时，激活的 PEG 的反应半衰期约为 5 小时。通过增加蛋白质孵育时间和浓度，可使颗粒表面上的抗体最大负载量达到 1.6 x 10(-2)个分子/纳米(2)。使用多重多组分光学编码的物种特异性免疫分析来评估测定的 S/N 比。编码的颗粒通过共价接枝或非特异性涂层与牛或鼠 IgG 结合，用于同时检测互补的抗 IgG“靶标”或不互补的抗 IgG“噪声”。通过将多克隆抗体或工程单链可变片段（scFv）捕获探针固定在颗粒上来检测卵巢癌标志物间皮素（MSLN），证明了其作为基于血清的测定平台的多功能性和潜力。MSLN 抗原被掺入 PBS 缓冲液或 50%人血清中。两种捕获探针方向和介质条件均显示出相似的低水平检测限为 5 ng/mL；然而，在 50%血清中运行的测定中，最大信号强度下降了 40%。
