Dynamic monitoring of a bi-enzymatic reaction at a single biomimetic giant vesicle

Lefrançois, Pauline; Goudeau, Bertrand; Arbault, Stéphane

doi:10.1039/d0an01273d

Cited by 9 publications

(13 citation statements)

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“…We assume that the flux detected at one location of the membrane is representative of the mean flux produced by the GUV. The hypothesis is reinforced by our recent results with the same type of enzymatic GUVs analyzed by fluorescence confocal microscopy . The Amplex Red assay was used to image the bienzymatic system composed of GOx and horseradish peroxidase (HRP); we observed that resorufin, the end-product of the reactions, was emitted over the whole surface of the GUV, meaning that the composition within the GUV volume was homogeneous at the timescale of the experiment.…”

Section: Resultsmentioning

confidence: 77%

“…The enzymatic reaction in single GUVs was induced by microinjections of low quantities of glucose oxidase (GOx from Aspergillus Niger). In a recent study, 73 we have defined the experimental conditions allowing a kinetic and quantitative control of the GOx activity in the same GUVs as used herein. A hydrogen peroxide fluorescent assay (Amplex Red fluorogenic probe and peroxidase enzyme) revealed by laser scanning confocal microscopy on the GUVs that such liposomes allow a passive diffusion of glucose and resorufin (fluorescent reporter of the Amplex Red assay).…”

Section: Performing the Gox Catalysis In A Single Guv And Detecting T...mentioning

confidence: 99%

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

2021

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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.

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Section: Resultsmentioning

confidence: 77%

Section: Performing the Gox Catalysis In A Single Guv And Detecting T...mentioning

confidence: 99%

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

2021

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“…The cascade reaction was mediated by two enzymes: glucose oxidase (GOx) and horseradish peroxidase (HRP), which work in tandem to oxidize a fluorogenic probe (Amplex TM Red). [43] The bioreactors were created by encapsulation of the GOx and HRP in giant polymer vesicles using the SDE method. The reaction was started by addition of Amplex TM Red into the medium surrounding the bioreactors to a final concentration of 30 µM.…”

Section: Resultsmentioning

confidence: 99%

Formation of giant polymer vesicles by simple double emulsification using block copolymers as the sole surfactant

et al. 2021

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“…In contrast, Lobovkina’s group reported that by applying positive pressure toward the inside of the GUV at the contact tip of a micropipette, a string-bead structure made of a stretched GV membrane was produced inside the GUV [ 81 ]. In addition, microinjection is also possible with a micropipette by piercing a part of the GV membrane with a tip with a diameter of 1 μm [ 82 , 83 , 84 ].…”

Section: Manipulation Methods Of Gvsmentioning

confidence: 99%

Identifying and Manipulating Giant Vesicles: Review of Recent Approaches

Toyota

Zhang

2022

Micromachines

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Giant vesicles (GVs) are closed bilayer membranes that primarily comprise amphiphiles with diameters of more than 1 μm. Compared with regular vesicles (several tens of nanometers in size), GVs are of greater scientific interest as model cell membranes and protocells because of their structure and size, which are similar to those of biological systems. Biopolymers and nano-/microparticles can be encapsulated in GVs at high concentrations, and their application as artificial cell bodies has piqued interest. It is essential to develop methods for investigating and manipulating the properties of GVs toward engineering applications. In this review, we discuss current improvements in microscopy, micromanipulation, and microfabrication technologies for progress in GV identification and engineering tools. Combined with the advancement of GV preparation technologies, these technological advancements can aid the development of artificial cell systems such as alternative tissues and GV-based chemical signal processing systems.

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Dynamic monitoring of a bi-enzymatic reaction at a single biomimetic giant vesicle

Abstract: Giant unilamellar vesicles were used as individual biomimetic micro-reactors wherein a model bi-enzymatic reaction involving a glucose oxidase (GOx) and the horseradish peroxidase (HRP) was monitored by confocal microscopy. These...

Cited by 9 publications

References 43 publications

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

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

Formation of giant polymer vesicles by simple double emulsification using block copolymers as the sole surfactant

Identifying and Manipulating Giant Vesicles: Review of Recent Approaches

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