Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA.
Anal Bioanal Chem. 2013 Apr;405(11):3831-8. doi: 10.1007/s00216-012-6656-5. Epub 2013 Jan 11.
We describe an electrochemiluminescence (ECL) immunoarray incorporated into a prototype microfluidic device for highly sensitive protein detection and apply this system to accurate, sensitive measurements of prostate-specific antigen (PSA) and interleukin-6 (IL-6) in serum. The microfluidic system employed three molded polydimethylsiloxane (PDMS) channels on a conductive pyrolytic graphite chip (2.5 × 2.5 cm) inserted into a machined chamber and interfaced with a pump, switching valve, and sample injector. Each of the three PDMS channels encompasses three 3 μL analytical wells. Capture-antibody-decorated single-wall carbon nanotube forests are fabricated in the bottom of the wells. The antigen is captured by these antibodies on the well bottoms. Then, a RuBPY-silica-secondary antibody (Ab2) label is injected to bind to antigen on the array, followed by injection of sacrificial reductant tripropylamine (TPrA) to produce ECL. For detection, the chip is placed into an open-top ECL measuring cell, and the channels are in contact with electrolyte in the chamber. Potential applied at 0.95 V versus Ag/AgCl oxidizes TPrA to produce ECL by redox cycling the RuBPY species in the particles, and ECL light is measured by a charge-coupled device camera. This approach achieved ultralow detection limits of 100 fg mL(-1) for PSA (9 zeptomole) and 10 fg mL(-1) (1 zeptomole) for IL-6 in calf serum, a 10-25-fold improvement of a similar non-microfluidic array. PSA and IL-6 in synthetic cancer patient serum samples were detected in 1.1 h and results correlated well with single-protein enzyme-linked immunosorbent assays.
我们描述了一种电化学发光（ECL）免疫阵列，该阵列集成到一个原型微流控设备中，用于高度灵敏的蛋白质检测，并将该系统应用于血清中前列腺特异性抗原（PSA）和白细胞介素-6（IL-6）的精确、灵敏测量。微流控系统在导电热解石墨芯片（2.5 × 2.5 cm）上使用三个模制的聚二甲基硅氧烷（PDMS）通道，插入加工腔中，并与泵、开关阀和样品注射器接口。三个 PDMS 通道中的每一个都包含三个 3 μL 的分析井。捕获抗体修饰的单壁碳纳米管森林在井的底部制造。抗原被这些抗体在井底部捕获。然后，注入 RuBPY-二氧化硅-二级抗体（Ab2）标记物以结合阵列上的抗原，然后注入牺牲还原剂三丙胺（TPrA）以产生 ECL。为了检测，将芯片放入顶部开口的 ECL 测量池中，通道与腔室中的电解质接触。在 0.95 V 相对于 Ag/AgCl 的电势下施加电势，通过在颗粒中氧化循环 RuBPY 物质来氧化 TPrA 以产生 ECL，并且通过电荷耦合器件相机测量 ECL 光。这种方法在小牛血清中实现了超低的检测限，PSA（9 飞摩尔）为 100 fg mL(-1)，IL-6 为 10 fg mL(-1)（1 飞摩尔），与类似的非微流控阵列相比提高了 10-25 倍。在合成癌症患者血清样本中检测到 PSA 和 IL-6，耗时 1.1 小时，结果与单蛋白酶联免疫吸附测定相关性良好。
