Key Laboratory of Analysis and Detection for Food Safety (MOE & Fujian Province), Department of Chemistry, Fuzhou University , Fuzhou 350108, People's Republic of China.
Anal Chem. 2013 Nov 5;85(21):10589-96. doi: 10.1021/ac402713a. Epub 2013 Oct 11.
A new fluorescence immunoassay strategy based on a target-induced displacement reaction with cargo release from protein-gated carbohydrate-functionalized magnetic mesoporous silica nanoparticles (MMSN) was developed for sensitive detection of small molecular mycotoxins (aflatoxin B1, AFB1 used in this case). To construct such an assay system, MMSN was initially functionalized with mannose-terminated silanes, then capped with biotinylated concanavalin A (Con A) entrapped rhodamine B (RB) within the pores through the carbohydrate-protein interaction, and then biotinylated monoclonal anti-AFB1 capture antibody was conjugated to Con A-functionalized MMSN by the streptavidin-biotin chemistry. Gold nanoparticles (AuNP) heavily functionalized with invertase and bovine serum albumin-AFB1 conjugate were utilized as the trace tag. With AFB1 introduction, a competitive immunoreaction for the immobilized anti-AFB1 antibody on the MMSN was started between target analyte and the labeled AFB1 on the AuNP. Accompanied by AuNP, the carried invertase hydrolyzed sucrose in glucose and fructose. The generated glucose competed with the mannose for Con A and displaced the Con A-antibody complex from the MMSN, resulting in the opening of molecular gates owing to the uncapping of MMSN, thereby the entrapped RB could release from the pores. The released RB could be quantitatively determined by a fluorometer. Under optimal conditions, the fluorescence intensity decreased with the increasing AFB1 concentration in the range from 0.01 to 5 ng mL(-1) with a detection limit (LOD) of 8 pg mL(-1) at the 3sblank criterion. Intra- and interbatch assay precisions were lower than 9 and 9.5% (CV), respectively. The method featured unbiased identification of negative (blank) and positive samples. No significant differences at the 0.05 significance level were encountered in the analysis of naturally contaminated peanut samples between the fluorescence immunoassay and a commercialized enzyme-linked immunosorbent assay (ELISA) method.
基于目标诱导的位移反应，从蛋白门控的糖基化磁性介孔硅纳米粒子（MMSN）中释放货物，开发了一种新的荧光免疫分析策略，用于灵敏检测小分子真菌毒素（本实验中使用黄曲霉毒素 B1，AFB1）。为了构建这样的分析系统，首先用末端为甘露糖的硅烷对 MMSN 进行功能化，然后通过碳水化合物-蛋白质相互作用将生物素化伴刀豆球蛋白 A（Con A）包封在孔内的罗丹明 B（RB）固定在 MMSN 上，然后通过链霉亲和素-生物素化学将生物素化的单克隆抗 AFB1 捕获抗体连接到 Con A 功能化的 MMSN 上。大量功能化的葡萄糖苷酶和牛血清白蛋白- AFB1 缀合物的金纳米粒子（AuNP）被用作示踪标记。随着 AFB1 的引入，目标分析物与 AuNP 上标记的 AFB1 之间在固定在 MMSN 上的抗 AFB1 抗体上开始了竞争性免疫反应。伴随着 AuNP，携带的葡萄糖苷酶在葡萄糖和果糖中将蔗糖水解。生成的葡萄糖与甘露糖竞争 Con A，并将 Con A-抗体复合物从 MMSN 上置换下来，由于 MMSN 的解盖帽，分子门打开，从而 RB 从孔中释放出来。通过荧光计可以定量测定释放的 RB。在最佳条件下，荧光强度随 AFB1 浓度在 0.01 至 5ng mL(-1) 范围内的增加而降低，检测限（LOD）在 3sblank 标准下为 8pg mL(-1)。批内和批间测定的精密度均低于 9%和 9.5%（CV）。该方法具有对阴性（空白）和阳性样品进行无偏识别的特点。在对天然污染花生样品的分析中，荧光免疫分析与商业化的酶联免疫吸附分析（ELISA）方法之间在 0.05 显著性水平上没有遇到显著差异。
