文獻(xiàn)：使用光鑷雕刻和融合仿生囊泡網(wǎng)絡(luò)

鏈接：https://www.nature.com/articles/s41467-018-04282-w

作者：吉多·博洛涅西馬克·S·弗里丁阿里·薩利希-雷哈尼內(nèi)森·E·巴洛，尼古拉斯·J·布魯克斯，奧斯卡·塞斯尤瓦爾·埃拉尼

節(jié)選：

囊泡融合和囊泡微反應(yīng)器

為了制備功能化的金納米粒子囊泡，我們首先在含有2 wt.% 16:0 生物素帽聚乙烯 (Biotinyl Cap PE) 的0.5 M蔗糖溶液中電鑄POPC囊泡。當(dāng)存在熒光脂質(zhì)時(shí)，其含量為1 wt.%。在鈣黃綠素稀釋和蛋白質(zhì)表達(dá)實(shí)驗(yàn)中，囊泡通過乳液相轉(zhuǎn)移（見上文）形成，濃度為0.5 M蔗糖/葡萄糖密度梯度。接下來，將150 nm鏈霉親和素包被的金納米粒子（Nanopartz，美國科羅拉多州；產(chǎn)品C11-150-TS-50；2.5 mg ml ?1）以1:9的比例添加到囊泡中，將樣品渦旋30分鐘以促進(jìn)結(jié)合。VIM的形成方法與之前相同，但外部溶液中的NaCl含量為0.25 M。通過在VIM界面上聚焦并施加150 mW（在阱處）或更高功率的激光來實(shí)現(xiàn)囊泡的融合。為了避免意外融合，使用小于100 mW的激光（在阱處）對囊泡進(jìn)行空間操控（補(bǔ)充說明 13）。我們還使用了Aurora-DSG納米粒子（AuNP標(biāo)記的脂質(zhì)；1-2 nm；Avanti），當(dāng)其以2 wt.%的濃度嵌入膜中時(shí)，不會發(fā)生融合。

Vesicle fusion and vesicle microreactors

To prepare functionalised AuNP vesicles, we first electroformed POPC vesicles in 0.5?M sucrose solution with 2 wt.% 16:0 Biotinyl Cap PE. When fluorescent lipids were present, these were at 1 wt.%. In the calcein dilution and protein expression experiments, vesicles were formed via emulsion phase transfer (see above), with 0.5?M sucrose/glucose density gradient. Next, 150?nm streptavidin-coated AuNPs (Nanopartz, CO, USA; product C11-150-TS-50; 2.5?mg ml?1) were added to the vesicles 1:9, with the sample vortexed for 30?min to drive conjugation. VIMs were formed as before, but with 0.25?M NaCl in the external solution. Vesicles were fused by focusing and applying a laser of 150?mW (at trap) or greater on the VIM interface. Vesicles were manipulated in space using a <100?mW laser (at trap) to avoid unintentional fusion (Supplementary Note 13). We also used Aurora-DSG Nanoparticles (AuNP-labelled lipids; 1–2?nm; Avanti), which did not result in fusion when embedded in the membrane at 2 wt.%.

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