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-1β 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.

光学免疫测定法小型化的最大挑战之一是微通道/微毛细管的光路距离短。蛋白质生物标志物经常出现在皮摩尔级至飞摩尔级的循环血液中，需要超高水平的灵敏度，而传统的生色底物和复杂的检测系统无法满足这种要求。这项研究基于增强酶促扩增，公开了一种有效的策略，可以提高光学询问的微流体装置中夹心ELISA的灵敏度并缩短总测试时间。我们发现辣根过氧化物酶（HRP）在中流系统中的活性受到扩散的高度限制，因此，在这些系统中增加酶促底物的浓度不会转化为酶促对话的增强。相反，由于扩散距离短，在微流体系统中会发生相反的情况，但是第二种酶促底物过氧化氢（H₂ O ₂），导致如本文报道的酶抑制。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小型化系统。含氟聚合物微流控条中癌症生物标志物PSA和人类细胞因子IL-1β的夹心ELISA定量分析显示，仅改变OPD：H ₂ O ₂的摩尔比，灵敏度可增加一个数量级，孵育时间可减少10倍从1：3至1：1，OPD浓度从1至4 mg / ml增加。酶促扩增的这种增强最终提供了利用廉价且普遍存在的智能手机和平板扫描仪对新型便携式和负担得起的微流体设备进行光学询问所需的灵敏度。