Phillips K Scott, Han Jong Ho, Cheng Quan
Department of Chemistry, University of California, Riverside, California 92521, USA.
Anal Chem. 2007 Feb 1;79(3):899-907. doi: 10.1021/ac0612426.
Detection of trace amounts of target proteins in the presence of high concentrations of matrix proteins (e.g., serum samples) without separation steps is of great significance to biomedical research but remains technically challenging. Here we report a "membrane cloaking" method to overcome nonspecific protein adsorption and fouling problems for label-free surface plasmon resonance detection and heterogeneous immunosensing. A thin, hybrid, self-assembled monolayer on gold was formed with 70 mol % mercaptopropanol and 30 mol % cysteamine/propanedithiol to facilitate membrane fusion and covalent attachment of antibodies. After antibody immobilization, the surface was incubated with lipid vesicles, which fused to form a supported membrane. The analyte spiked in serum was introduced for binding, and the membrane and nonspecifically adsorbed proteins on the membrane were subsequently removed using a nonionic surfactant before the final measurement was carried out. Selection of a suitable surfactant can preserve antibody/antigen binding and selectively remove the membrane, allowing accurate measurement of the captured proteins without interference from nonspecifically adsorbed species. Surface plasmon resonance (SPR) quantification of IgG spiked in undiluted serum ( approximately 75 mg/mL protein) was achieved with the membrane cloaking method, whereas direct measurement without membrane removal resulted in a significantly large error. The cloaking method was also used to develop an enzyme amplified amperometric assay using HRP-conjugated IgG. Detection of concentrations as low as 5 fM proteins was obtained. Finally, a membrane cloaking assay combining SPR and in situ electrochemical measurement was demonstrated on a gold substrate. Similar sensitivity was observed using a continuous flow injection measurement. The method opens new avenues to develop direct assay methods with ultrahigh sensitivity for protein samples using SPR and enzyme-linked amplification mechanisms.
在不进行分离步骤的情况下,在高浓度基质蛋白(如血清样本)存在时检测痕量目标蛋白,对生物医学研究具有重要意义,但在技术上仍具有挑战性。在此,我们报告一种“膜掩盖”方法,以克服无标记表面等离子体共振检测和异质免疫传感中的非特异性蛋白质吸附和污染问题。用70摩尔%的巯基丙醇和30摩尔%的半胱胺/丙二硫醇在金表面形成一层薄的混合自组装单层,以促进膜融合和抗体的共价连接。抗体固定后,将表面与脂质囊泡孵育,脂质囊泡融合形成支撑膜。引入加标在血清中的分析物进行结合,随后在进行最终测量之前,使用非离子表面活性剂去除膜和膜上非特异性吸附的蛋白质。选择合适的表面活性剂可以保留抗体/抗原结合,并选择性地去除膜,从而在不受非特异性吸附物质干扰的情况下准确测量捕获的蛋白质。通过膜掩盖方法实现了对加标在未稀释血清(约75mg/mL蛋白质)中的IgG的表面等离子体共振(SPR)定量,而不去除膜的直接测量导致显著较大的误差。该掩盖方法还用于开发一种使用辣根过氧化物酶(HRP)偶联IgG的酶放大安培测定法。获得了低至5fM蛋白质浓度的检测结果。最后,在金基底上展示了一种结合SPR和原位电化学测量的膜掩盖测定法。使用连续流动注射测量观察到了类似的灵敏度。该方法为利用SPR和酶联放大机制开发对蛋白质样品具有超高灵敏度的直接测定方法开辟了新途径。