Electroanalysis at a Single Giant Vesicle Generating Enzymatically a Reactive Oxygen Species,Analytical Chemistry

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Electroanalysis at a Single Giant Vesicle Generating Enzymatically a Reactive Oxygen Species
Analytical Chemistry ( IF 6.7 ) Pub Date : 2021-09-21 , DOI: 10.1021/acs.analchem.1c01208
Pauline Lefrançois ₁ , Jérôme Santolini ₂ , Stéphane Arbault _{1,

3}

Affiliation

In the framework of artificial or synthetic cell development, giant liposomes are common basic structures. Their enclosed membrane allows encapsulating proteins, DNA, reactants, etc., while its phospholipid nature allows some exchanges with the surrounding medium. Biochemical reactions induced inside giant liposomes or vesicles are often monitored or imaged by fluorescence microscopy techniques. Here, we show that electrochemistry performed with ultramicroelectrodes is perfectly suitable to monitor an enzymatic reaction occurring in a single giant unilamellar vesicle. Glucose oxidase (GOx) was microinjected inside individual vesicles containing 1 mM glucose. H₂O₂ was thus generated in the vesicle and progressively diffused across the membrane toward the surrounding environment. An ultramicroelectrode sensitive to H₂O₂ (black platinum-modified carbon surface) was placed next to the membrane and provided a direct detection of the hydrogen peroxide flux generated by the enzyme activity. Electrochemistry offered a highly sensitive (in situ detection), selective (potential applied at the electrode), time-resolved analysis (chronoamperometry) of the GOx activity over an hour duration, without modifying the internal giant unilamellar vesicles (GUV) medium. These results demonstrate that electroanalysis with microsensors is well adapted and complementary to fluorescence microscopy to sense enzymatic activities, for instance, generating reactive oxygen species, at single vesicles further used to develop artificial cells.

中文翻译：

单个巨型囊泡的电分析酶促反应性氧物种

在人工或合成细胞发育的框架中，巨型脂质体是常见的基本结构。它们的封闭膜允许封装蛋白质、DNA、反应物等，而其磷脂性质允许与周围介质进行一些交换。在巨大的脂质体或囊泡内诱导的生化反应通常通过荧光显微镜技术进行监测或成像。在这里，我们表明使用超微电极进行的电化学非常适合监测单个巨大单层囊泡中发生的酶促反应。葡萄糖氧化酶 (GOx) 被显微注射到含有 1 mM 葡萄糖的单个囊泡内。H ₂ O ₂因此在囊泡中产生并逐渐通过膜向周围环境扩散。对 H ₂ O ₂（黑色铂改性碳表面）敏感的超微电极放置在膜旁边，并提供对酶活性产生的过氧化氢通量的直接检测。电化学提供了一种高度灵敏的（原位检测）、选择性（施加在电极上的电位）、时间分辨分析（计时电流法）一小时内的 GOx 活动，而无需修改内部巨型单层囊泡 (GUV) 介质。这些结果表明，微传感器的电分析非常适合并与荧光显微镜互补，以感知酶活性，例如，在进一步用于开发人工细胞的单个囊泡中产生活性氧。

更新日期：2021-10-06

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