Graduate School of Engineering, Kobe University, 1-1, Kobe 657-8501, Japan.
Biosens Bioelectron. 2013 May 15;43:45-9. doi: 10.1016/j.bios.2012.11.021. Epub 2012 Dec 3.
Simple method to immobilize antibody in organic/inorganic hybrid thin films, prepared easily by using liquid phase deposition (LPD), was proposed to enhance the sensitivity, reproducibility and stability of immunosensing chips. The LPD method could embed a monoclonal antibody of C-reactive protein (Anti-CRP), which is mixed with poly(l-lysine) (PL) as an organic binder, on/in titanium oxide thin layer deposited onto a surface plasmon resonance (SPR) sensing chip (F(Anti-CRP-PL)), allowing simple immobilization of anti-CRP capable of CRP detection. As a control, anti-CRP was non-covalently immobilized after preparing the titanium oxide layer containing PL (SIF(Anti-CRP-PL)). Binding experiments of CRP on the prepared sensor chips were performed by SPR measurements. The CRP binding capacity of anti-CRP on F(Anti-CRP-PL) was 54% greater than that of anti-CRP on SIF (Anti-CRP-PL), suggesting that the immobilization method could retain good antigen-binding activity of anti-CRP. Reproducibility of the antigen-antibody interaction was estimated by SPR using 10mM HCl as the regeneration regent. CRP was repeatedly detected, and the detection limit was calculated to be 19.9ngmL(-1) (CV: 8.9%). By using this method, antibodies could be simply immobilized to various kinds of sensing chips for the development of immunosensing systems.
本研究提出了一种简单的方法，通过液相沉积（LPD）制备有机/无机杂化薄膜，将抗体固定在其中，以提高免疫传感芯片的灵敏度、重现性和稳定性。该方法可以将 C 反应蛋白（CRP）的单克隆抗体（Anti-CRP）嵌入到聚赖氨酸（PL）作为有机结合物中，然后将其沉积到表面等离子体共振（SPR）传感芯片上的氧化钛薄膜中（F（Anti-CRP-PL）），从而实现简单固定 Anti-CRP，用于 CRP 的检测。作为对照，在制备含有 PL 的氧化钛层后，非共价固定 Anti-CRP（SIF（Anti-CRP-PL））。通过 SPR 测量对制备好的传感器芯片进行 CRP 与抗体的结合实验。结果表明，F（Anti-CRP-PL）上 Anti-CRP 的 CRP 结合能力比 SIF（Anti-CRP-PL）上的 Anti-CRP 高 54%，表明该固定方法可以保留 Anti-CRP 良好的抗原结合活性。使用 10mM HCl 作为再生试剂，通过 SPR 评估抗原-抗体相互作用的重现性。可以对 CRP 进行重复检测，检测限计算为 19.9ngmL(-1)（CV：8.9%）。通过使用这种方法，可以将抗体简单地固定到各种传感芯片上，用于开发免疫传感系统。
