Biosciences Institute, The Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Orla Protein Technologies Ltd., Biosciences Centre, International Centre for Life, Times Square, Newcastle upon Tyne NE1 4EP, UK.
Int J Mol Sci. 2023 Apr 20;24(8):7599. doi: 10.3390/ijms24087599.
Surface plasmon resonance (SPR) is a very sensitive measure of biomolecular interactions but is generally too expensive for routine analysis of clinical samples. Here we demonstrate the simplified formation of virus-detecting gold nanoparticle (AuNP) assemblies on glass using only aqueous buffers at room temperature. The AuNP assembled on silanized glass and displayed a distinctive absorbance peak due to the localized SPR (LSPR) response of the AuNPs. Next, assembly of a protein engineering scaffold was followed using LSPR and a sensitive neutron reflectometry approach, which measured the formation and structure of the biological layer on the spherical AuNP. Finally, the assembly and function of an artificial flu sensor layer consisting of an in vitro-selected single-chain antibody (scFv)-membrane protein fusion was followed using the LSPR response of AuNPs within glass capillaries. In vitro selection avoids the need for separate animal-derived antibodies and allows for the rapid production of low-cost sensor proteins. This work demonstrates a simple approach to forming oriented arrays of protein sensors on nanostructured surfaces that uses (i) an easily assembled AuNP silane layer, (ii) self-assembly of an oriented protein layer on AuNPs, and (iii) simple highly specific artificial receptor proteins.
表面等离子体共振(SPR)是一种非常灵敏的生物分子相互作用测量方法,但通常由于成本过高,无法用于临床样本的常规分析。在这里,我们展示了仅使用室温下的水性缓冲液在玻璃上简化形成用于检测病毒的金纳米粒子(AuNP)组装体的方法。AuNP 在硅烷化玻璃上组装,并由于 AuNP 的局域表面等离子体共振(LSPR)响应而显示出独特的吸光度峰。接下来,使用 LSPR 和灵敏的中子反射率方法跟踪蛋白质工程支架的组装,该方法测量了球形 AuNP 上生物层的形成和结构。最后,使用玻璃毛细管内的 AuNP 的 LSPR 响应,跟踪由体外选择的单链抗体(scFv)-膜蛋白融合体组成的人工流感传感器层的组装和功能。体外选择避免了对单独的动物衍生抗体的需求,并允许快速生产低成本的传感器蛋白。这项工作展示了一种在纳米结构表面上形成定向蛋白传感器阵列的简单方法,该方法使用(i)易于组装的 AuNP 硅烷层,(ii)AuNP 上定向蛋白层的自组装,以及(iii)简单的高特异性人工受体蛋白。