Multifunctional Dendrimer-templated Antibody Presentation on Biosensor Surfaces for Improved Biomarker Detection.
Multifunctional Dendrimer-templated Antibody Presentation on Biosensor Surfaces for Improved Biomarker Detection.
复制标题
DOI：
10.1002/adfm.200901293
复制
发表时间：
2010-02-08
影响因子：
19
通讯作者：
Romero R
中科院分区：
文献类型：
--
作者：
Han HJ;Kannan RM;Wang S;Mao G;Kusanovic JP;Romero R
树枝状聚合物具有明确的球状形状和高密度的官能团，是用于模板化传感器表面的理想纳米级材料。这项工作利用树枝状大分子作为捕获生物标志物的多功能平台，并提高灵敏度和特异性。描述了基于树枝状聚合物的测定平台的合成、表征、制造和功能验证。合成双功能羟基/硫醇官能化 G4-聚酰胺胺 (PAMAM) 树枝状聚合物，并通过偶联 PEG-马来酰亚胺和树枝状聚合物硫醇基团将其固定在聚乙二醇 (PEG) 功能化测定板上。同时，部分树枝状聚合物硫醇基团转化为酰肼官能团。所得树状聚合物修饰表面与氧化抗体 Fc 区的捕获抗体偶联。这保留了抗体抗原结合区 (Fv) 的方向灵活性。为了验证该方法，制作的板进一步用作开发夹心型 ELISA 的固相，以检测 IL-6 和 IL-1β（绒毛膜羊膜炎早期阶段的重要生物标志物）。树枝状聚合物修饰板的检测灵敏度显着增强，非特异性吸附更低，IL-6鲁米诺检测的检测限为0.13 pg ml-1，IL-1β TMB检测的检测限为1.15 pg ml-1，明显优于传统ELISA。这些测定在正常（非怀孕）女性和患有肾盂肾炎的孕妇的人血清样本中得到了验证。基于树枝状聚合物的捕获策略的特异性和改进的灵敏度可能对多种细胞因子和生物标志物的检测具有重要意义，因为捕获策略可应用于多重微珠测定、电导免疫传感器和场效应生物传感器。
Dendrimers, with their well-defined globular shape and a high density of functional groups, are ideal nanoscale materials for templating sensor surfaces. This work exploits dendrimers as a versatile platform for capturing biomarkers with improved sensitivity and specificity. Synthesis, characterization, fabrication, and functional validation of the dendrimer-based assay platform are described. Bifunctional hydroxyl/thiol functionalized G4-polyamidoamine (PAMAM) dendrimer is synthesized and immobilized on to the polyethylene-glycol (PEG)-functionalized assay plate by coupling PEG-maleimide and dendrimer thiol groups. Simultaneously, part of the dendrimer thiol groups are converted to hydrazide functionalities. The resulting dendrimer-modified surface is coupled to the capture antibody in the Fc region of the oxidized antibody. This preserves the orientation flexibility of the antigen binding region (Fv) of the antibody. To validate the approach, the fabricated plates are further used as a solid phase for developing a sandwich type ELISA to detect IL-6 and IL-1β, important biomarkers for early stages of chorioamnionitis. The dendrimer-modified plate provides assays with significantly enhanced sensitivity, lower nonspecific adsorption, and a detection limit of 0.13 pg ml-1 for IL-6 luminol detection and 1.15 pg ml-1 for IL-1β TMB detection, which are significantly better than those for the traditional ELISA. The assays were validated in human serum samples from normal (non-pregnant) woman and pregnant women with pyelonephritis. The specificity and the improved sensitivity of the dendrimer-based capture strategy could have significant implications for the detection of a wide range of cytokines and biomarkers since the capture strategy could be applied to multiplex microbead assays, conductometric immunosensors and field effect biosensors.