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.

中文翻译：

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用于细胞外钾检测的基于囊泡的传感器

介绍

能够原位检测生物离子的传感器的设计仍然具有挑战性。虽然存在许多可以提供快速、轻松读数的荧光指示剂，但它们通常是非特异性的，特别是对于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，该传感器利用膜通道将钾 (K ⁺ ) 离子门控到封装的荧光指示剂。

方法

我们组装了包含缬氨霉素（一种 K ⁺特异性膜转运蛋白）和封装间苯二甲酸苯并呋喃 (PBFI) 的磷脂囊泡，它是一种在其他离子（如钠 (Na ⁺ )存在下非特异性发出荧光的 K ⁺敏感染料）。封装的 PBFI 荧光的特异性、动力学和可逆性在酶标仪和荧光计中确定。然后将传感器添加到大肠杆菌细菌培养物中，以评估培养基中的 K ⁺水平作为细胞密度的函数。

结果

与染料在溶液中游离的对照相比，囊泡传感器在存在竞争性单价离子钠 (Na ⁺ ) 和二价阳离子钙 (Ca ²⁺ ) 的情况下显着提高了 K ^{+检测的特异性。}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平台，该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。通过改变膜转运蛋白和封装传感器，我们的方法应该广泛用于设计生物传感器，以在传统上难以监测的环境中检测一系列生物分析物。