Biomedical Research Institute , Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Langmuir. 2013 Oct 22;29(42):13111-20. doi: 10.1021/la402802u. Epub 2013 Oct 9.
In order to develop a biosensing system with waveguide-mode sensor, fabrication of a biosensing interface on the silica surface of the sensing chip was carried out using triethoxysilane derivatives with anti-leptin antibody. Triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties were synthesized to immobilize the antibody and to suppress nonspecific adsorption of proteins, respectively. The chip modified with triethoxysilane derivatives bearing oligoethylene glycol moiety suppressed nonspecific adsorption of proteins derived from human serum effectively by rinse with PBS containing surfactant (0.05% Tween 20). On the other hand, it was confirmed that antibody was immobilized on the chip by immersion into antibody solution to show response of antigen-antibody reaction, where the chip was modified with triethoxysilane derivatives bearing succinimide ester moiety. When the interface was fabricated with antibody and a mixture of triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties, the response of antigen-antibody reaction depended on composition of the mixture and enhanced with the increase of ratio for triethoxysilane derivatives bearing succinimide ester moiety reflecting the antibody concentration immobilized on the chip. While introduction of excess triethoxysilane derivatives bearing succinimide ester moiety induced nonspecific adsorption of proteins derived from human serum, the immobilized antibody on the chip kept its activity after 1-month storage in a refrigerator. Taking into consideration those factors, the biosensing interface was fabricated using triethoxysilane derivatives with anti-leptin antibody to examine performance of the waveguide-mode sensor. It was found that the detection limits for human leptin were 50 ng/mL in PBS and 100 ng/mL in human serum. The results demonstrate that the waveguide-mode sensor powered by the biosensing interface fabricated with those triethoxysilane derivatives and antibody has potential to detect several tens of nanograms per milliliter of biomarkers in human serum with an unlabeled detection method.
为了开发基于波导模式传感器的生物传感系统，在传感芯片的二氧化硅表面上使用带有抗瘦素抗体的三乙氧基硅烷衍生物进行了生物传感界面的制备。分别合成了带有琥珀酰亚胺酯和聚乙二醇基团的三乙氧基硅烷衍生物，以固定抗体并抑制蛋白质的非特异性吸附。用含有表面活性剂（0.05%吐温 20）的 PBS 冲洗修饰有带有聚乙二醇基团的三乙氧基硅烷衍生物的芯片可有效抑制人血清中蛋白质的非特异性吸附。另一方面，通过将芯片浸入抗体溶液中以显示抗原-抗体反应的响应来证实抗体固定在芯片上，其中芯片用带有琥珀酰亚胺酯基团的三乙氧基硅烷衍生物修饰。当界面用抗体和带有琥珀酰亚胺酯和聚乙二醇基团的三乙氧基硅烷衍生物的混合物制备时，抗原-抗体反应的响应取决于混合物的组成，并随着固定在芯片上的抗体浓度的增加而增强。虽然引入过量的带有琥珀酰亚胺酯基团的三乙氧基硅烷衍生物会导致人血清中蛋白质的非特异性吸附，但在冰箱中储存 1 个月后，固定在芯片上的抗体仍保持其活性。考虑到这些因素，使用带有抗瘦素抗体的三乙氧基硅烷衍生物制备了生物传感界面，以检查波导模式传感器的性能。结果发现，在 PBS 中对人瘦素的检测限为 50ng/mL，在人血清中为 100ng/mL。结果表明，用这些三乙氧基硅烷衍生物和抗体制备的生物传感界面为动力的波导模式传感器具有以非标记检测方法检测人血清中数十纳克/毫升生物标志物的潜力。
