Genetically Encoded FRET-Based Nanosensor for Real-Time Monitoring of A549 Exosomes: Early Diagnosis of Cancer

微泡 费斯特共振能量转移 化学 A549电池 外体 细胞生物学 癌细胞 生物标志物 共焦显微镜 纳米传感器 生物物理学 癌症研究 纳米技术 癌症 小RNA 荧光 体外 生物化学 生物 基因 材料科学 量子力学 物理 遗传学
作者
Reshma Bano,Neha Soleja,Mohd. Mohsin
出处
期刊:Analytical Chemistry [American Chemical Society]
卷期号:95 (13): 5738-5746 被引量:13
标识
DOI:10.1021/acs.analchem.2c05774
摘要

Exosomes contain a plethora of unique disease biomarkers involving cellular homeostasis, infection dissemination, cancer development, and cardiac diseases. Exosomes originating from cancer cells have promising biomarkers for the early detection and assessment of the therapeutic response to cancer. The exosomal epidermal growth factor receptor (EGFR) is a potential biomarker which is overexpressed in cancer; thus, the level of EGFR expression is investigated by so many methods in a liquid and solid biopsy. The optimal method for isolating pure exosomal EGFRs has not been well understood so far. Current approaches are complicated and time-consuming, therefore hampering their clinical applications. Here, we demonstrate the creation of an innovative fluorescence resonance energy transfer (FRET) sensor, named ExoSen (exosome sensor), which can be implemented to determine the concentration of exosomal EGFRs at in vitro as well as in vivo levels. In this study, a sensing element for A549 exosomes, mitogen-inducible gene 6 (MIG6), has been employed between the FRET pair ECFP and Venus. MIG6 binding to ExoSen induced a conformational change that can be monitored by a variation in the FRET ratio. Moreover, the developed sensor, expressed in bacterial, yeast, and HEK-293T cells, demonstrates an increased FRET ratio with the addition of A549 exosomes, which can quantify the A549 exosomes noninvasively. The ExoSen enables rapid detection of A549 exosomes with great sensitivity at a concentration of 3.5 × 109 particles/mL. ExoSen is stable to pH fluctuations and provides a highly accurate, real-time optical readout in cell-based experiments by using confocal microscopy.
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