Functional screening of intracellular proteins in single cells and in patterned cell arrays using electroporation
Functional screening of intracellular proteins in single cells and in patterned cell arrays using electroporation
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DOI：
10.1021/ac025584x
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发表时间：
2002-08-15
影响因子：
7.4
通讯作者：
Orwar, O
中科院分区：
文献类型：
--
作者：
Nolkrantz, K;Farre, C;Orwar, O
提出了一种用于检测和表征单个靶细胞或小细胞群细胞质内的细胞内酶-底物和受体-配体相互作用的工具。荧光酶底物和受体配体通过电渗快速递送，并使用高电压偏压的充满电解质的毛细管（EFC）通过电穿孔在细胞中内化。具体而言，分别使用二磷酸荧光素和酪蛋白 BODIPY FL 在单个 NG108-15 细胞中检测碱性磷酸酶和蛋白酶。 EFC 引入选择性受体激动剂 IP3 和环腺苷二磷酸核糖 (cADPr) 后，分别检测细胞内 1,4,5-肌醇三磷酸 (IP3) 和兰尼碱受体。在这两种情况下，受体激活都会导致胞质中游离钙离子的浓度增加，这是使用钙离子选择性探针 Fluo-3 进行测量的。 cADPr 的作用可以通过与兰尼定受体拮抗剂钌红共同给药来阻断。此外，还演示了在PDMS中模制的微孔结构（100×100×45μm）中生长的多个细胞的电穿孔。使用 EFC 进行电穿孔和传递蛋白质标记、配体和底物的方法和系统可能可用于细胞内靶标的高通量筛选，并应用于蛋白质组学和表型分析以及药物发现。
A tool for detection and characterization of intracellular enzyme-substrate and receptor-ligand interactions inside the cytoplasm of single targeted cells or small confined groups of cells is presented. Fluorogenic enzyme substrates and receptor ligands were rapidly delivered by electroosmosis and internalized by electroporation in cells using an electrolyte-filled capillary (EFC) biased at a high voltage. Specifically, alkaline phosphatase and proteases were detected in single NG108-15 cells using fluorescein diphosphate and casein BODIPY FL, respectively. The intracellular 1,4,5-inositol triphosphate (IP3) and ryanodine receptors were detected after EFC introduction of the selective receptor agonists IP3 and cyclic adenosine diphosphate ribose (cADPr), respectively. Receptor activation in both cases resulted in increased cytosolic concentrations of free calcium ions that were measured using the calcium-ion-selective probe, fluo-3. The effect of cADPr could be blocked by coadministration of the ryanodine receptor antagonist ruthenium red. Furthermore, electroporation of a plurality of cells grown in microwell structures (100 x 100 x 45 mum) molded in PDMS is demonstrated. The methods and systems described using an EFC for electroporation and delivery of protein markers, ligands, and substrates might be useful in high-throughput screening of intracellular targets, with applications in proteomics and phenotype profiling, as well as in drug discovery.