Graduate School of Material Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako, Hyogo 678-1297, Japan.
Biosens Bioelectron. 2011 Oct 15;28(1):443-9. doi: 10.1016/j.bios.2011.07.073. Epub 2011 Aug 4.
In this study, a rapid immunosensing system has been developed for simultaneous analysis of two tumor markers, alpha-fetoprotein (AFP) and prostate-specific antigen (PSA). The strategy for rapid multisensing is based on rapid immunoreactions occurring on the surface of microparticles and the spatial separation of different particles that exhibit distinct dielectrophoretic (DEP) properties. Recognition events for immunoreactions have been performed on the surfaces of two different microparticles conjugated with two different antibodies: polystyrene (PS) microparticles with an anti-AFP antibody and gold-coated (50 nm) PS microparticles with an anti-PSA antibody. The DEP devices consisted of an upper indium tin oxide (ITO) glass and a lower ITO electrode with a castellated structure. Sandwich structured immunocomplexes of AFP and PSA were created on the microparticles and then labeled with fluorescent molecules via a secondary antibody. After introducing the particles into the DEP devices, an alternating current (AC) voltage (20 V peak-to-peak voltage and 30 kHz) was applied between the upper ITO and lower electrodes to manipulate the particles with negative dielectrophoresis (n-DEP).The uncoated PS particles and the gold-coated PS particles rapidly moved and separated to form wave-like line and triangular aggregates, respectively. The measurements of the fluorescence signals from the uncoated and gold-coated PS particles directed to different regions of the DEP device permit the determination of the concentrations of AFP and PSA simultaneously. No cross-reactivity was observed for either of the immunorecognition events. Limits of detection achieved for the AFP and PSA assays were 0.18 and 1.1 ng mL(-1), respectively, which satisfy medical requirements for both antigens in human serum. The total assay time required for the simultaneous detection of the two different analytes in this study (25 min) was shortened compared to the conventional enzyme-linked immunosorbent assay.
在这项研究中，开发了一种快速免疫传感系统，用于同时分析两种肿瘤标志物，甲胎蛋白（AFP）和前列腺特异性抗原（PSA）。快速多传感策略基于在微粒表面上发生的快速免疫反应以及表现出不同介电泳（DEP）特性的不同粒子的空间分离。免疫反应的识别事件已在两种不同的与两种不同抗体偶联的微粒表面上进行：与抗 AFP 抗体偶联的聚苯乙烯（PS）微粒和与抗 PSA 抗体偶联的金涂覆（50nm）PS 微粒。DEP 器件由上铟锡氧化物（ITO）玻璃和具有 castellated 结构的下 ITO 电极组成。AFP 和 PSA 的夹心结构免疫复合物在微粒上形成，然后通过二级抗体用荧光分子标记。将粒子引入 DEP 器件后，在上 ITO 和下电极之间施加交流（AC）电压（20V 峰峰值和 30kHz），以通过负介电泳（n-DEP）操纵粒子。未涂覆的 PS 粒子和金涂覆的 PS 粒子快速移动并分离，分别形成波状线和三角形聚集体。对指向 DEP 器件不同区域的未涂覆 PS 粒子和金涂覆 PS 粒子的荧光信号的测量允许同时确定 AFP 和 PSA 的浓度。两种免疫识别事件均未观察到交叉反应。AFP 和 PSA 测定的检测限分别为 0.18 和 1.1ngmL(-1)，分别满足人血清中两种抗原的医学要求。与传统的酶联免疫吸附测定相比，本研究中同时检测两种不同分析物所需的总测定时间（25 分钟）缩短了。
