Magnetic Bead-Based Reverse Colorimetric Immunoassay Strategy for Sensing Biomolecules
Magnetic Bead-Based Reverse Colorimetric Immunoassay Strategy for Sensing Biomolecules
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基于磁珠的反比色免疫分析策略用于传感生物分子
DOI：
10.1021/ac401433p
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发表时间：
2013-07-16
影响因子：
7.4
通讯作者：
Tang, Dianping
中科院分区：
文献类型：
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作者：
Gao, Zhuangqiang;Xu, Mingdi;Tang, Dianping
通过将高催化效率的过氧化氢酶与基于磁珠的过氧化物酶模拟物偶联，首次设计并使用了一种新颖的反比色免疫分析（RCIA）策略，用于灵敏检测生物体液中的低丰度蛋白质（本例中使用的前列腺特异性抗原，PSA）。为了构建这样的 RCIA 系统，首先合成了包括磁珠和金纳米颗粒的两种纳米结构，并分别用抗 PSA 捕获抗体和过氧化氢酶/抗 PSA 检测抗体进行功能化。此后，通过使用功能性金纳米粒子作为酶生物反应器和抗PSA缀合磁珠作为量热显影剂，采用特定的夹心型免疫测定形式来测定PSA。携带的过氧化氢酶以及夹心的免疫复合物部分消耗了检测溶液中添加的过氧化氢，从而减慢了基于磁珠的过氧化物酶模拟物对TMB/H2O2的催化效率，从而削弱了可见颜色并降低了比色密度。与传统的量热免疫分析不同，RCIA方法测定消耗后底物中残留的过氧化氢。在最佳条件下，所开发的RC1A对PSA表现出0.05-20 ng mL(-1)的宽动态范围，在3S（空白）水平下的检测限为0.03 ng mL(-1)。分析内和分析间变异系数分别低于 6.1% 和 9.3%。此外，该方法还针对 12 份 PSA 临床血清样本的分析进行了进一步验证，结果与市售酶联免疫吸附测定 (ELISA) 方法获得的结果非常一致。
A novel reverse colorimetric immunoassay (RCIA) strategy was for the first time designed and utilized for sensitive detection of low-abundance protein (prostate-specific antigen, PSA, used in this case) in biological fluids by coupling highly catalytic efficient catalase with magnetic bead-based peroxidase mimics. To construct such a RCIA system, two nanostructures including magnetic beads and gold nanoparticles were first synthesized and functionalized with anti-PSA capture antibody and catalase/anti-PSA detection antibody, respectively. Thereafter, a specific sandwich-type immunoassay format was employed for determination of PSA by using functional gold nanopartides as enzymatic bioreactors and anti-PSA-conjugated magnetic beads as a calorimetric developer. The carried catalase, followed by the sandwiched immunocomplex, partially consumed the added hydrogen peroxide in the detection solution, which slowed down the catalytic efficiency of magnetic bead-based peroxidase mimics toward TMB/H2O2, thereby weakening the visible color and decreasing the colorimetric density. Different from conventional calorimetric immunoassay, the RCIA method determined the residual hydrogen peroxide in the substrate after consumption. Under the optimal conditions, the developed RC1A exhibited a wide dynamic range of 0.05-20 ng mL(-1) toward PSA with a detection limit of 0.03 ng mL(-1) at the 3S(blank) level. Infra- and interassay coefficients of variation were below 6.1% and 9.3%, respectively. Additionally, the methodology was further validated for the analysis of 12 PSA clinical serum specimens, giving results in good accordance with those obtained by the commercially available enzyme-linked immunosorbent assay (ELISA) method.