Masumoto Mayu, Ohta Sera, Nakagawa Misaki, Hiruta Yuki, Citterio Daniel
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan.
Anal Bioanal Chem. 2022 Jan;414(1):691-701. doi: 10.1007/s00216-021-03682-0. Epub 2021 Oct 18.
This manuscript reports on a simple paper-based bienzymatic colorimetric assay for sarcosine as an important urinary biomarker of prostate cancer. All required assay reagents are pre-deposited on hydrophilic filter paper spots surrounded by a hydrophobic barrier. Sarcosine in the sample solution is selectively oxidized in the presence of sarcosine oxidase (SOx), resulting in the formation of hydrogen peroxide, which is subsequently detected through the horseradish peroxidase (HRP)-catalyzed conversion of the colorless indicator 3,3',5,5'-tetramethylbenzidine (TMB) into its blue-colored oxidation product. By the modification of the paper with positively charged poly(allylamine hydrochloride) (PAH), a linear response to sarcosine between 0 and 10 μM and a significant lowering of the limit of detection (LOD) (0.6 μM) compared to the unmodified paper substrate (12.6 μM) has been achieved. The improvement of the LOD was attributed to the fact that the presence of the polymer limits the enzyme-driven colorimetric reaction to the surface of the paper substrate, resulting in stronger color development. In experiments in artificial urine matrix, the bicarbonate anion was identified as an inhibitor of the colorimetric reaction. This inhibition was successfully eliminated through on-device sample pH adjustments with pH-buffer components pre-deposited onto assay devices. The LOD for sarcosine achieved in artificial urine matrix (2.5 μM) is below the 5 μM threshold value for this urinary biomarker required for diagnostic purposes. Finally, good selectivity over all 20 natural amino acids and satisfactory long-term storage stability of reagent-modified paper substrates at - 20 °C for a period of 50 days were confirmed.
本手稿报道了一种简单的基于纸的双酶比色法，用于检测肌氨酸，肌氨酸是前列腺癌的一种重要尿液生物标志物。所有所需的检测试剂预先沉积在由疏水屏障包围的亲水性滤纸片上。样品溶液中的肌氨酸在肌氨酸氧化酶（SOx）存在下被选择性氧化，生成过氧化氢，随后通过辣根过氧化物酶（HRP）催化将无色指示剂3,3',5,5'-四甲基联苯胺（TMB）转化为蓝色氧化产物来进行检测。通过用带正电荷的聚（烯丙胺盐酸盐）（PAH）对纸张进行改性，与未改性的纸质底物（12.6 μM）相比，实现了对0至10 μM肌氨酸的线性响应以及检测限（LOD）的显著降低（0.6 μM）。检测限的提高归因于聚合物的存在将酶驱动的比色反应限制在纸质底物表面，从而导致更强的显色效果。在人工尿液基质实验中，碳酸氢根阴离子被确定为比色反应的抑制剂。通过使用预先沉积在检测装置上的pH缓冲成分在装置上对样品pH进行调节，成功消除了这种抑制作用。在人工尿液基质中实现的肌氨酸检测限（2.5 μM）低于该尿液生物标志物用于诊断目的所需的5 μM阈值。最后，证实了对所有20种天然氨基酸具有良好的选择性，并且试剂改性的纸质底物在-20°C下具有50天的令人满意的长期储存稳定性。
