Cao Yong, McDermott Mark T
Department of Chemistry, 11227 Saskatchewan Drive, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada; National Research Council Canada, Nanotechnology Research Centre (NRC NANO), 11421 Saskatchewan Drive, Edmonton, Alberta, T6G 2M9, Canada.
Department of Chemistry, 11227 Saskatchewan Drive, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada; National Research Council Canada, Nanotechnology Research Centre (NRC NANO), 11421 Saskatchewan Drive, Edmonton, Alberta, T6G 2M9, Canada.
Anal Biochem. 2018 Sep 15;557:7-12. doi: 10.1016/j.ab.2018.06.027. Epub 2018 Jun 28.
Quantitative measurement of small-molecule metabolites is now emerging as an effective way to link the metabolite profile to disease state. Surface plasmon resonance (SPR) is a sensing platform that has demonstrated applicability for a large range of biomolecules. However, direct detection of small molecules with SPR challenges the refractive index based detection mechanism. Herein, we utilized an indirect detection format and developed an inhibition immunoassay for the quantitative measurement of 17β-estradiol (E2) using SPR. One competitor, BSA-E2 conjugate, was immobilized to the SPR chip via the reaction between the primary amino group of the conjugate and the succinimide group (NHS) introduced by the formation of a thiol-NHS monolayer on gold surface. Free E2 molecules compete with BSA-E2 on chip surface for binding sites provided by a monoclonal anti-E2 antibody. It was found the binding affinity of the antibody to BSA-E2 conjugate increases with decreasing surface coverage of BSA-E2 conjugate. Under optimal conditions, a sigmoidal calibration curve with a negative slope and a dynamic range from 10 pM to 2 nM was generated. The detection limit of the immunoassay is estimated to be 0.3 pM. Moreover, the immunoassay exhibits high specificity for E2 detection using estrone (E1) as a potential interference.
小分子代谢物的定量测量正逐渐成为一种将代谢物谱与疾病状态联系起来的有效方法。表面等离子体共振（SPR）是一种传感平台，已证明适用于多种生物分子。然而，用SPR直接检测小分子对基于折射率的检测机制提出了挑战。在此，我们采用间接检测形式，开发了一种基于SPR的抑制免疫分析法，用于定量测量17β-雌二醇（E2）。一种竞争物，即牛血清白蛋白-E2偶联物，通过偶联物的伯氨基与金表面硫醇-NHS单层形成引入的琥珀酰亚胺基团（NHS）之间的反应固定在SPR芯片上。游离的E2分子与芯片表面的牛血清白蛋白-E2竞争单克隆抗E2抗体提供的结合位点。发现抗体与牛血清白蛋白-E2偶联物的结合亲和力随着牛血清白蛋白-E2偶联物表面覆盖率的降低而增加。在最佳条件下，生成了具有负斜率且动态范围为10 pM至2 nM的S形校准曲线。该免疫分析法的检测限估计为0.3 pM。此外，该免疫分析法以雌酮（E1）作为潜在干扰物时，对E2检测具有高特异性。
