Department of Science of Technology Innovation, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan.
Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan.
Anal Chem. 2022 Feb 8;94(5):2416-2424. doi: 10.1021/acs.analchem.1c04108. Epub 2021 Dec 28.
Droplet-based microfluidic systems are a powerful tool for biological assays with high throughput. Water-in-oil droplets (WODLs) are typically used in droplet-based microfluidic systems to culture microorganisms and perform enzyme assays. However, because of the oil surrounding the nanoliter and picoliter volumes of WODLs, availability of suitable substrates is limited. For instance, although 7-amino-4-methylcoumarin (AMC) is commonly used as a fluorescent probe of the substrate to detect peptidase activity, AMC leaks from WODLs to the oil phase due to its high hydrophobicity. Thus, AMC substrates cannot be used in droplet-based microfluidic systems with WODLs. In this study, we developed a peptidase substrate consisting of a dipeptide and 7-aminocoumarin-4-acetic acid (ACA), an AMC-derived fluorogenic compound. ACA was retained in the WODL for more than 7 days, and the dipeptidyl ACA substrate detected dipeptidyl peptidase (DPP) activity in the WODL. Compared to AMC substrates, the substrate specificity constants of DPPs for ACA substrates increased up to 4.7-fold. Fluorescence-activated droplet sorting made high-throughput screening of microorganisms based on DPP activity using the dipeptidyl ACA substrate possible. Since ACA could be applied to various substrates as a fluorescent probe, detectable microbial enzyme activities for droplet-based microfluidic systems can be largely expanded.
基于液滴的微流控系统是一种高通量生物分析的有力工具。油包水液滴（WODLs）通常用于基于液滴的微流控系统中培养微生物和进行酶分析。然而，由于 WODLs 周围的油相，合适的底物可用性受到限制。例如，虽然 7-氨基-4-甲基香豆素（AMC）通常用作检测肽酶活性的底物的荧光探针，但由于其高疏水性，AMC 会从 WODLs 泄漏到油相中。因此，AMC 底物不能用于基于液滴的微流控系统中的 WODLs。在这项研究中，我们开发了一种由二肽和 7-氨基香豆素-4-乙酸（ACA）组成的肽酶底物，ACA 是一种 AMC 衍生的荧光化合物。ACA 在 WODL 中保留了超过 7 天，并且二肽酰基 ACA 底物可以检测 WODL 中的二肽酶（DPP）活性。与 AMC 底物相比，DPPs 对 ACA 底物的特异性常数增加了高达 4.7 倍。荧光激活的液滴分选使得基于 DPP 活性的微生物高通量筛选成为可能，使用二肽酰基 ACA 底物。由于 ACA 可以作为荧光探针应用于各种底物，因此可以大大扩展基于液滴的微流控系统中可检测的微生物酶活性。
