Electroanalysis at a Single Giant Vesicle Generating Enzymatically a Reactive Oxygen Species

Abstract

In the framework of artificial or synthetic cell development, giant liposomes are common basic structures. Their enclosed membrane permits to encapsulate 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 micro-injected 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 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. can be easily manipulated individually and observed by optical microscopies (fluorescence microscopy in particular). Consequently, GUVs offer an excellent platform to study the enzymatic generation of ROS or RNS in minimal cells.

Recent advances in single liposome electrochemistry

“…The GUV size allows to combine electrochemical and optical microscopy measurements such as chronoamperometry coupled to fluorescence in order to reach a high level of sensitivity and analysis of the vesicle content and its environment. In addition, contrary to living cells, the stability and easy preparation of GUVs are attractive advantages allowing to modulate the lipid membrane composition and the microinjection through it for detecting electrochemical reactions at the single vesicle scale [16,54]. The electroanalysis of a single GUV is usually performed by chronoamperometry recorded onto an UME polarized at the potential of the vesicle electrochemical content and placed in the vicinity of the GUV, close to the lipid membrane [16].…”

“…In addition, contrary to living cells, the stability and easy preparation of GUVs are attractive advantages allowing to modulate the lipid membrane composition and the microinjection through it for detecting electrochemical reactions at the single vesicle scale [16,54]. The electroanalysis of a single GUV is usually performed by chronoamperometry recorded onto an UME polarized at the potential of the vesicle electrochemical content and placed in the vicinity of the GUV, close to the lipid membrane [16]. Because of the relative high permeability of the lipid membrane, the vesicle opening and the content release / electrolysis are fast mechanisms, which give the opportunity to encapsulate a large variety of different probes and to quickly analyse biochemical reactions induced following the injection (micropipette) of different species inside the GUV [16,54].…”

“…The electroanalysis of a single GUV is usually performed by chronoamperometry recorded onto an UME polarized at the potential of the vesicle electrochemical content and placed in the vicinity of the GUV, close to the lipid membrane [16]. Because of the relative high permeability of the lipid membrane, the vesicle opening and the content release / electrolysis are fast mechanisms, which give the opportunity to encapsulate a large variety of different probes and to quickly analyse biochemical reactions induced following the injection (micropipette) of different species inside the GUV [16,54]. Thanks to the size of GUV and their ability to encapsulate a highly reactive-load, the electrochemical signal recorded at the polarized UME corresponding to the content release / electrolysis is significantly and accurately detected (several pico-amperes) [16].…”

“…Because of the relative high permeability of the lipid membrane, the vesicle opening and the content release / electrolysis are fast mechanisms, which give the opportunity to encapsulate a large variety of different probes and to quickly analyse biochemical reactions induced following the injection (micropipette) of different species inside the GUV [16,54]. Thanks to the size of GUV and their ability to encapsulate a highly reactive-load, the electrochemical signal recorded at the polarized UME corresponding to the content release / electrolysis is significantly and accurately detected (several pico-amperes) [16]. The main advantages of the electroanalysis of single GUV are probably the ability to make versatile micro-reactors with various contents allowing a high electrochemical sensing and to simultaneously combine with optical microscopy measurements.…”

“…In the present review, we will focus on the recent advances in single liposome electrochemistry including single-vesicle electrochemical events with the exocytosis process [11][12][13], electrochemical single impacts of synthetic redox liposomes [7,14,15], and the electroanalysis of single giant unilamellar vesicle (GUV) [16,17]. We will present these three different electroanalytical techniques and discuss their current limitations and applications through different recent examples.…”