Instituto de Química , Universidade Federal de Goiás , Campus Samambaia , Goiânia , Goiás 74690-900 , Brazil.
Embrapa Agroenergia , Empresa Brasileira de Pesquisa Agropecuária , Brasília , Distrito Federal 70770-901 , Brazil.
Anal Chem. 2018 Oct 16;90(20):11949-11954. doi: 10.1021/acs.analchem.8b02384. Epub 2018 Sep 24.
This study describes the use of mass spectrometry imaging with matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) to understand the color gradient generation commonly seen in microfluidic paper-based analytical devices (μPADs). The formation of color gradients significantly impacts assay sensitivity and reproducibility with μPADs but the mechanism for formation is poorly understood. The glucose enzymatic assay using potassium iodide (KI) as a chromogenic agent was selected to investigate the color gradient generated across a detection spot. Colorimetric measurements revealed that the relative standard deviation for the recorded pixel intensities ranged between 34 and 40%, compromising the analytical reliability. While a variety of hypotheses have been generated to explain this phenomenon, few studies have attempted to elucidate the mechanisms associated with its formation. Mass spectrometry imaging using MALDI and DESI was applied to understand the nonuniform color distribution on the detection zone. MALDI experiments were first explored to monitor the spatial distribution of the glucose oxidase and horseradish peroxidase mixture, before and after lateral flow assay with and without KI. MALDI(+)-TOF data revealed uniform enzyme distribution on the detection spots. On the other hand, after the complete assay DESI(-) measurements revealed a heterogeneous shape indicating the presence of iodide and triiodide ions at the zone edge. The reaction product (I) is transported by lateral flow toward the zone edge, generating the color gradient. Mass spectrometry imaging has been used for the first time to prove that color gradient forms as result of the mobility small molecules and not the enzyme distribution on μPAD surface.
本研究采用基质辅助激光解吸/电离（MALDI）和解吸电喷雾电离（DESI）质谱成像技术，研究了微流控纸基分析器件（μPAD）中常见的颜色梯度形成。颜色梯度的形成显著影响μPAD 分析的灵敏度和重现性，但形成机制尚不清楚。本研究选择使用碘化钾（KI）作为显色剂的葡萄糖酶促测定来研究检测点处产生的颜色梯度。比色测量结果表明，记录像素强度的相对标准偏差在 34%至 40%之间，这影响了分析的可靠性。虽然已经提出了各种假设来解释这种现象，但很少有研究试图阐明与其形成相关的机制。本研究采用 MALDI 和 DESI 质谱成像来了解检测区域上不均匀的颜色分布。首先探索 MALDI 实验以监测葡萄糖氧化酶和辣根过氧化物酶混合物在侧向流动分析前后的空间分布，有无 KI 存在时均进行了检测。MALDI（+）-TOF 数据显示检测点上的酶分布均匀。另一方面，在完成整个测定后，DESI（-）测量结果显示出不均匀的形状，表明在区域边缘存在碘离子和三碘化物离子。反应产物（I）通过侧向流向区域边缘迁移，从而产生颜色梯度。本研究首次使用质谱成像技术证明，颜色梯度的形成是由于小分子的迁移，而不是酶在μPAD 表面的分布。
