Chair for Analytical Chemistry and Institute of Hydrochemistry, Technische Universität München, Marchioninistr. 17, Munich, Germany.
Sorbonne Universités, UPMC Univ Paris 6, UMR CNRS 7197, Laboratoire de Réactivité de Surface, F75005 Paris, France; CNRS, UMR 7197, Laboratoire de Réactivité de Surface, F75005 Paris, France.
Biosens Bioelectron. 2015 May 15;67:334-41. doi: 10.1016/j.bios.2014.08.047. Epub 2014 Aug 23.
Small molecules (haptens) like pharmaceuticals or peptides can serve as targets for antibody binding in competitive immunoassay-based flow-through assays. In this work, a strategy for preparing polyethylene glycol (PEG) coatings for subsequent hapten immobilization on glass-type silica surfaces is presented and characterized in detail. Two substrates bearing terminal silanol groups were utilized, a glass slide and a silicon wafer. First, surfaces were thoroughly cleaned and pretreated to generate additional silanol groups. Then, a silane layer with terminal epoxy groups was created using 3-glycidyloxypropyltrimethoxysilane (GOPTS). Epoxy groups were used to bind a layer of diamino-poly(ethylene glycol) (DAPEG) with terminal amino groups. Finally, the low molecular weight compound diclofenac was bound to the surface to be used as model ligand for competitive biosensing of haptens. The elementary steps were characterized using atomic force microscopy (AFM), water contact angle measurement, grazing-angle attenuated total reflection (GA-ATR) FT-IR spectroscopy, and X-ray photoelectron spectroscopy (XPS). The data collected using these techniques have confirmed the successive grafting of the molecular species, evidencing, that homogeneous monolayers were created on the silica surfaces and validated the proposed mechanism of functionalization. The resulting surfaces were used to investigate polyclonal anti-diclofenac antibodies recognition and reversibility using quartz crystal microbalance with dissipation (QCM-D) measurements or an automated flow-through immunoassay with chemiluminescence (CL) read-out. For both techniques, recognition and reversibility of the antibody binding were observed. The stability of sensors over time was also assessed and no decrease in CL response was observed upon 14 days in aqueous solution. The herein presented strategy for surface functionalization can be used in the future as reproducible and reusable universal platform for hapten biosensors.
小分子（半抗原）如药物或肽可以作为抗体结合的靶标，用于基于竞争免疫测定的流动通过测定。在这项工作中，提出并详细表征了一种用于在玻璃型硅胶表面上随后固定半抗原的聚乙二醇（PEG）涂层的制备策略。使用了两种带有末端硅醇基团的基底，即玻片和硅片。首先，彻底清洁和预处理表面以生成额外的硅醇基团。然后，使用 3-缩水甘油氧基丙基三甲氧基硅烷（GOPTS）生成带有末端环氧基团的硅烷层。环氧基团用于结合带有末端氨基的二氨基聚（乙二醇）（DAPEG）层。最后，将低分子量化合物双氯芬酸结合到表面上，用作竞争生物传感半抗原的模型配体。使用原子力显微镜（AFM）、水接触角测量、掠角衰减全反射（GA-ATR）FT-IR 光谱和 X 射线光电子能谱（XPS）对基本步骤进行了表征。使用这些技术收集的数据证实了分子物种的连续接枝，证明了在硅胶表面上形成了均匀的单层，并验证了所提出的功能化机制。使用石英晶体微天平（QCM-D）测量或带有化学发光（CL）读出的自动流动通过免疫测定来研究多克隆抗双氯芬酸抗体的识别和可逆性。对于这两种技术，都观察到了抗体结合的识别和可逆性。还评估了传感器随时间的稳定性，在水溶液中 14 天后，CL 响应没有下降。所提出的表面功能化策略可作为未来重现性和可重复使用的通用半抗原生物传感器平台。
