Sensitive optical detection of clinically relevant biomarkers in affordable microfluidic devices: Overcoming substrate diffusion limitations
Sensitive optical detection of clinically relevant biomarkers in affordable microfluidic devices: Overcoming substrate diffusion limitations
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DOI：
10.1016/j.snb.2017.11.086
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发表时间：
2018-04-01
影响因子：
8.4
通讯作者：
Reis, Nuno M.
中科院分区：
文献类型：
--
作者：
Barbosa, Ana I.;Castanheira, Ana P.;Reis, Nuno M.
光学免疫分析小型化的最大挑战之一是微通道/微毛细管的光路距离较短。蛋白质生物标志物通常存在于血液中的微微摩尔-微微摩尔范围内，需要特殊的灵敏度，而传统的显色底物和没有复杂、庞大的检测系统都无法满足这一要求。本研究提出了一种基于增强酶扩增的微流控器件夹心ELISA检测方法，提高了检测的灵敏度，缩短了检测时间。我们发现，辣根过氧化物酶(HRP)在中流体系中的活性受到扩散的高度限制，因此增加这些体系中酶底物的浓度并不能转化为酶转化的增强。在微流控系统中，由于扩散距离较短，情况正好相反，然而，如本文所报道的，第二种酶底物过氧化氢(H_2O_2)浓度的增加会导致酶抑制。因此，我们发现商业底物配方中邻苯二胺(OPD)与过氧化氢的摩尔比不适合于小型化系统。对含氟聚合物微流控试条中肿瘤生物标记物PSA和人类细胞因子IL-1β的夹心ELISA法定量显示，只要将OPD：H2O2的摩尔比从1：3改变到1：1，并将OPD的浓度从1 mg/ml增加到4 mg/ml，灵敏度就提高了一个数量级以上，孵育时间减少了10倍。这种酶扩增的增强最终提供了光学询问新型便携式和负担得起的微流控设备所需的灵敏度，这些设备配备了廉价和无处不在的智能手机和平板扫描仪。(C)2017爱思唯尔B.V.保留所有权利。
One of the biggest challenges in miniaturization of optical immunoassays is the short light path distance of microchannels/microcapillaries. Protein biomarkers are often presented in circulating blood in the picomolar-femtomolar range, requiring exceptional levels of sensitivity that cannot be met with traditional chromogenic substrates and without sophisticated, bulky detection systems. This study discloses an effective strategy for increasing the sensitivity and shorten the total test time for sandwich ELISAs in microfluidic devices optically interrogated, based on enhancing enzymatic amplification. We found that activity of Horseradish Peroxidase (HRP) in mesofluidic systems is highly limited by diffusion, therefore increasing the concentration of enzymatic substrate in these systems does not translate into an enhancement in enzymatic conversation. The opposite happens in microfluidic systems due to short diffusion distances, however increased concentration of the second enzymatic substrate, hydrogen peroxide (H2O2), leads to enzyme inhibition as herein reported. Consequently, we found that the molar ratio of o-phenylenediamine (OPD) to hydrogen peroxide from commercially substrate formulations is not suitable for miniaturized systems. Sandwich ELISA quantitation of a cancer biomarker PSA and human cytokine IL-1 beta in fluoropolymer microfluidic strips revealed over one order of magnitude increase in sensitivity and 10-fold decrease in incubation time by simply changing the molar ratio of OPD: H2O2 from 1: 3 to 1: 1 and increasing OPD concentration from 1 to 4 mg/ml. This enhancement in enzymatic amplification offers finally the sensitivity required for optical interrogation of novel portable and affordable microfluidic devices with inexpensive and ubiquitous smartphones and flatbed scanners. (C) 2017 Elsevier B.V. All rights reserved.