School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, PR China.
Biosens Bioelectron. 2011 Oct 15;28(1):421-7. doi: 10.1016/j.bios.2011.07.056. Epub 2011 Jul 30.
A "dual-layer membrane cloaking" (DLMC) method was developed to construct disposable electrochemical immunosensor for direct determination of serum sample. Mouse IgG (MIgG) molecules were firstly immobilized on a substrate. After the formation of a didodecyldimethylammonium bromide (DDAB) membrane on the MIgG modified substrate, an additional bovine serum albumin (BSA) thin layer was formed to build a BSA/DDAB dual-layer membrane (DLM). When alkaline phosphatase conjugated anti-mouse IgG antibodies (anti-MIgG-ALP) in human serum were incubated on the substrate, anti-MIgG-ALP was recognized specifically by the immobilized MIgG while all nonspecifically adsorbed proteins were selectively removed together with BSA/DDAB DLM by 5% Triton X-100 (v/v) before final measurements. The BSA/DDAB DLM was characterized and optimized by surface plasmon resonance (SPR) technique, and further employed in a disposable immunoassay based on an ITO chip. Under optimal conditions, MIgG in human serum was directly detected in the range of 2.0-18.0 ng mL(-1) without dilution or separation. A limit of detection as low as 0.922 ng mL(-1) (6.15 pM) was obtained. The proposed DLMC method can efficiently prevent the penetration of matrix proteins through single cloaking membrane and completely eliminate nonspecific adsorption. It has great potential in providing a versatile way for direct determination of serum sample with ultra-sensitivity.
一种“双层膜伪装”（DLMC）方法被开发出来，用于构建一次性电化学免疫传感器，以直接测定血清样品。首先将鼠 IgG（MIgG）分子固定在基底上。在 MIgG 修饰基底上形成双十二烷基二甲基溴化铵（DDAB）膜之后，形成额外的牛血清白蛋白（BSA）薄层以构建 BSA/DDAB 双层膜（DLM）。当人血清中的碱性磷酸酶共轭抗鼠 IgG 抗体（抗-MIgG-ALP）孵育在基底上时，抗-MIgG-ALP 特异性地被固定的 MIgG 识别，而所有非特异性吸附的蛋白质则与 BSA/DDAB DLM 一起通过 5%Triton X-100（v/v）选择性地去除，然后进行最终测量。BSA/DDAB DLM 通过表面等离子体共振（SPR）技术进行了表征和优化，并进一步用于基于 ITO 芯片的一次性免疫测定。在最佳条件下，人血清中的 MIgG 可以在未经稀释或分离的情况下直接在 2.0-18.0ng mL(-1)范围内检测到。检测限低至 0.922ng mL(-1)（6.15pM）。所提出的 DLMC 方法可以有效地防止基质蛋白通过单层伪装膜的渗透，并完全消除非特异性吸附。它在提供超灵敏的直接测定血清样品的通用方法方面具有很大的潜力。
