FRET-based fluorescent nanoprobe platform for sorting of active microorganisms by functional properties
FRET-based fluorescent nanoprobe platform for sorting of active microorganisms by functional properties
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基于 FRET 的荧光纳米探针平台，用于按功能特性对活性微生物进行分类
DOI：
10.1016/j.bios.2019.111832
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发表时间：
2020-01-15
影响因子：
12.6
通讯作者：
Sun, Guoping
中科院分区：
文献类型：
--
作者：
Luo, Yeshen;Liu, Fei;Sun, Guoping
荧光激活细胞分选术（FACS）很少应用于微生物筛选，因为检测分辨率较差，而稳定性较差、毒性或所用荧光传感器背景荧光的干扰会影响检测分辨率。在这里，首先使用荧光共振能量转移（FRET）荧光纳米探针NP-RA开发了基于荧光的快速高通量细胞分选方法，所述荧光纳米探针NP-RA通过用罗丹明B和甲基红（偶氮染料）涂覆二氧化硅纳米颗粒而构建。罗丹明B（内层）是FRET供体，甲基红（外层）是受体。这种即用型NPRA是非荧光的，但一旦外层被微生物降解就会发出荧光。在我们的实验中，NPRA对模式菌株脱色希瓦氏菌S12超灵敏，在共聚焦激光扫描显微镜下显示出8.0 cfu/mL至8.7 x 10（8）cfu/mL的宽检测范围，在荧光计下显示出1.1 x 10（7）至9.36 x 10（8）cfu/mL的宽检测范围。此外，NP-RA生物成像可以清楚地识别微生物群落中的其他偶氮呼吸细胞，包括Bosea thiooxidans DSM 9653和Lysinibacillus pakistanensis NCCP-54。此外，荧光探针NP-RA与下游FACS相容，使得可以直接从人工微生物群落中快速分选出偶氮呼吸细胞。据我们所知，还没有荧光纳米探针被设计用于跟踪和分选偶氮呼吸功能微生物。
Fluorescence-activated cell sorting (FACS) has rarely been applied to screening of microorganisms because of poor detection resolution, which is compromised by poor stability, toxicity, or interference from background fluorescence of the fluorescence sensors used. Here, a fluorescence-based rapid high-throughput cell sorting method was first developed using a fluorescence resonance energy transfer (FRET) fluorescent nanoprobe NP-RA, which was constructed by coating a silica nanoparticle with Rhodamine B and methyl-red (an azo dye). Rhodamine B (inner layer) is the FRET donor and methyl-red (outer layer) is the acceptor. This ready-to-use NPRA is non-fluorescent, but fluoresces once the outer layer is degraded by microorganisms. In our experiment, NPRA was ultrasensitive to model strain Shewanella decolorationis S12, showing a broad detection range from 8.0 cfu/mL to 8.7 x 10(8) cfu/mL under confocal laser scanning microscopy, and from 1.1 x 10(7) to 9.36 x 10(8) cfu/mL under a fluorometer. In addition, NP-RA bioimaging can clearly identify other azo-respiring cells in the microbial community, including Bosea thiooxidans DSM 9653 and Lysinibacillus pakistanensis NCCP-54. Furthermore, the fluorescent probe NP-RA is compatible with downstream FACS so that azo-respiring cells can be rapidly sorted out directly from an artificial microbial community. To our knowledge, no fluorescent nanoprobe has yet been designed for tracking and sorting azo-respiration functional microorganisms.