Vesicle-Based Sensors for Extracellular Potassium Detection
Vesicle-Based Sensors for Extracellular Potassium Detection
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DOI：
10.1007/s12195-021-00688-7
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发表时间：
2021-08-10
影响因子：
2.8
通讯作者：
Kamat, Neha P.
中科院分区：
文献类型：
--
作者：
Boyd, Margrethe A.;Davis, Anna M.;Kamat, Neha P.
能够原位检测生物离子的传感器的设计仍然具有挑战性。虽然存在许多荧光指示器，可以提供快速，容易的读数，但它们往往是非特异性的，特别是具有相似电荷状态的离子。为了解决这个问题，我们开发了一种基于囊泡的传感器，利用膜通道将钾离子（K+）通道连接到封装的荧光指示剂上。方法：我们组装了磷脂囊泡，其中包含了缬霉素（一种K+特异性膜转运蛋白）和包被苯并呋喃异眼酸盐（PBFI），一种K+敏感染料，在其他离子如钠（Na+）存在时非特异性荧光。用平板阅读器和荧光仪测定包封的PBFI荧光的特异性、动力学和可逆性。然后将传感器添加到大肠杆菌培养物中，以评估培养基中K+水平作为细胞密度的函数。结果相对于溶液中游离染料的对照，囊泡传感器在存在竞争性一价离子钠（Na+）和二价阳离子钙（Ca2+）时显著提高了K+检测的特异性。该传感器能够报告K+浓度的增加和减少。最后，我们观察到我们的囊泡传感器可以检测细菌培养物中K+浓度的变化。结论我们的数据为细胞外离子检测提供了一个新的平台，利用离子特异性膜转运蛋白来提高离子检测的特异性。通过改变膜转运体和封装传感器，我们的方法应该广泛适用于设计在传统上难以监测的环境中检测一系列生物分析物的生物传感器。
Introduction The design of sensors that can detect biological ions in situ remains challenging. While many fluorescent indicators exist that can provide a fast, easy readout, they are often nonspecific, particularly to ions with similar charge states. To address this issue, we developed a vesicle-based sensor that harnesses membrane channels to gate access of potassium (K+) ions to an encapsulated fluorescent indicator. Methods We assembled phospholipid vesicles that incorporated valinomycin, a K+ specific membrane transporter, and that encapsulated benzofuran isophthalate (PBFI), a K+ sensitive dye that nonspecifically fluoresces in the presence of other ions, like sodium (Na+). The specificity, kinetics, and reversibility of encapsulated PBFI fluorescence was determined in a plate reader and fluorimeter. The sensors were then added to E. coli bacterial cultures to evaluate K+ levels in media as a function of cell density. Results Vesicle sensors significantly improved specificity of K+ detection in the presence of a competing monovalent ion, sodium (Na+), and a divalent cation, calcium (Ca2+), relative to controls where the dye was free in solution. The sensor was able to report both increases and decreases in K+ concentration. Finally, we observed our vesicle sensors could detect changes in K+ concentration in bacterial cultures. Conclusion Our data present a new platform for extracellular ion detection that harnesses ion-specific membrane transporters to improve the specificity of ion detection. By changing the membrane transporter and encapsulated sensor, our approach should be broadly useful for designing biological sensors that detect an array of biological analytes in traditionally hard-to-monitor environments.