On-chip enzyme immunoassay of a cardiac marker using a microfluidic device combined with a portable surface plasmon resonance system
On-chip enzyme immunoassay of a cardiac marker using a microfluidic device combined with a portable surface plasmon resonance system
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DOI：
10.1021/ac060480y
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发表时间：
2006-08-01
影响因子：
7.4
通讯作者：
Niwa, Osamu
中科院分区：
文献类型：
--
作者：
Kurita, Ryoji;Yokota, Yoshimi;Niwa, Osamu
本文报道了一种微型免疫传感器，利用微流控装置与便携式表面等离子体共振（SPR）传感器系统相结合，用于检测微量水平的心脏标志物b型利钠肽（BNP）。将样品BNP溶液与乙酰胆碱酯酶标记的抗体（偶联物）免疫反应后引入微通道，在流动通道中只有未结合的偶联物被捕获在BNP固定的表面上。然后，将捕获的共轭物与酶促反应生成的硫醇化合物积累在位于微通道下游的金薄膜上，以实时监测SPR角位移。通过监测SPR角度位移，我们实现了5pg /mL- 100ng /mL的检测浓度范围，覆盖了血液中BNP浓度的检测范围。这一成功是由于使用了t形微流控装置结构，它可以防止样品溶液流过用于SPR检测的金膜。我们能够在30分钟内测量微量BNP肽（15 fg）的水平，因为我们的免疫传感器的程序比多步免疫分析更简单，通过同时使用标记的酶促反应和微流控装置中酶促产物积累的实时监测。我们采用该方法在人血清中使用加标样品检测血清BNP，并对经过热处理的样品在免疫反应前使血清中的酯酶变性获得满意的回收率。
This paper reports a miniaturized immunosensor designed to determine a trace level cardiac marker, B-type natriuretic peptide (BNP), using a microfluidic device combined with a portable surface plasmon resonance (SPR) sensor system. Sample BNP solution was introduced into the microchannel after an immunoreaction with acetylcholine esterase-labeled antibody (conjugate), and only unbound conjugate was trapped on the BNP-immobilized surface in the flow channel. Then, the thiol compound generated by the enzymatic reaction with the trapped conjugate was accumulated on a gold thin film located downstream in the microchannel to monitor the real-time SPR angle shift. We achieved a detectable concentration range of 5 pg/mL-100 ng/mL by monitoring the SPR angle shift, which covers the required detection range for the BNP concentrations found in blood. This success resulted from the use of a T-shaped microfluidic device structure, which prevents the sample solution from flowing over the gold film used for SPR detection. We were able to measure trace levels of BNP peptide (15 fg) within 30 min since the procedure with our immunosensor is simpler than a multistep immunoassay through the simultaneous use of a labeled enzymatic reaction and the real-time monitoring of enzymatic product accumulation in the microfluidic device. We employed the procedure to detect serum BNP by using spiked samples in human serum and achieved satisfactory recovery for heat-treated samples to denature the esterase in the serum before the immunoreaction.