BioAnalytical Chemistry and Nanobiomedicine Laboratory, Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan; Department of Chemistry, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Road, Hsinchu 30013, Taiwan.
BioAnalytical Chemistry and Nanobiomedicine Laboratory, Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan; Department of Chemistry, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Road, Hsinchu 30013, Taiwan.
Biosens Bioelectron. 2014 May 15;55:32-8. doi: 10.1016/j.bios.2013.10.054. Epub 2013 Dec 1.
Electrochemical method and surface plasmon resonance (SPR) spectroscopic analysis are utilized herein to investigate antibody immobilization without and with orientation control for site-positioning paratopes (antigen binding site) of the antibody molecules. Biotin and its antibody were selected in current study as model. Such an approach employed thiophene-3-boronic acid (T3BA) as paratope orientation controller, (i) enabled site orientation of the antibody molecules reducing the hiding of paratopes, and (ii) maintained the activity of the captured antibodies, as confirmed by electrochemical and SPR analysis. Anti-biotin antibody (a glycoprotein) was covalently bound to a self-assembled monolayer of T3BA modified on a nanogold-electrodeposited screen-printed electrode through boronic acid-saccharide interactions, with the boronic acid units specifically binding to the glycosylation sites of the antibody molecules. The immunosensor functioned based on competition between the analyte biotin and biotin-tagged, potassium hexacyanoferrate(II)-encapsulated liposomes. The current signal produced by the released liposomal Fe(CN)6(4-), measured using square wave voltammetry, yielded a sigmoidally shaped dose-response curve that was linear over eight orders of magnitude (from 10(-11) to 10(-3)M). Furthermore this biosensing system fabricated based on T3BA approach was found to possess significantly improved sensitivity, and the limit of detection toward biotin was calculated as 0.102 ng mL(-1) (equivalent to 6 μL of 4.19 × 10(-10)M biotin).
电化学方法和表面等离子体共振（SPR）光谱分析用于研究抗体的固定化，而无需和需要控制抗体分子的位定位变区（抗原结合位点）的取向。在本研究中选择生物素及其抗体作为模型。这种方法采用噻吩-3-硼酸（T3BA）作为变区取向控制器，（i）使抗体分子的位取向，减少变区的隐藏，和（ii）保持捕获抗体的活性，这通过电化学和 SPR 分析得到证实。抗生物素抗体（糖蛋白）通过硼酸-糖相互作用共价结合到纳米金电沉积的丝网印刷电极上的 T3BA 自组装单层上，硼酸单元特异性结合到抗体分子的糖基化位点上。免疫传感器基于分析物生物素和生物素标记的、包封亚铁氰化钾（II）的脂质体之间的竞争起作用。通过方波伏安法测量释放的脂质体 Fe(CN)6(4-)产生的电流信号，得到一个呈 S 形的剂量-反应曲线，在八个数量级（从 10(-11)到 10(-3)M）范围内呈线性。此外，基于 T3BA 方法制造的这种生物传感系统被发现具有显著提高的灵敏度，并且生物素的检测限计算为 0.102ng mL(-1)（相当于 4.19×10(-10)M 生物素的 6μL）。
