Dual-pore glass chips for cell-attached single-channel recordings.
Dual-pore glass chips for cell-attached single-channel recordings.
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用于细胞附着单通道记录的双孔玻璃芯片。
DOI：
10.1039/c4lc00370e
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发表时间：
2014
期刊：
影响因子：
6.1
通讯作者：
Mayer,Michael
中科院分区：
文献类型：
--
作者：
Bruhn,BrandonR;Liu,Haiyan;Schuhladen,Stefan;Hunt,AlanJ;Mordovanakis,Aghapi;Mayer,Michael
虽然现在已经建立了高通量平面膜片钳仪器来进行药物筛选的全细胞记录，但传统的基于微量移液器的方法仍然是进行细胞附着单通道记录的金标准。一般来说，平面平台不太适合这样的研究，由于过度的噪音导致低密封电阻和使用的基板介电性能差。由于这些平台倾向于使用相同的孔通过抽吸定位细胞并建立密封，因此来自细胞悬浮液的生物碎片可能在密封形成之前污染孔表面，从而降低密封阻力。在这里，飞秒激光烧蚀被用来制造双孔玻璃芯片优化用于细胞附着的单通道记录，通过使用不同的孔来定位细胞并建立密封来规避这个问题。这种双孔设计还允许使用相对较小的膜片孔径（D ~ 150至300 nm），这比平面膜片钳装置中通常使用的微孔（D ~ 1至2 μm）更适合于建立高阻力密封，而不会影响装置定位细胞的能力。利用玻璃基板实现的高密封电阻和低电容和介电噪声，使用这些双孔芯片的膜片钳实验始终实现高密封电阻（千兆密封形成率= 61%，平均密封电阻= 53 GΩ），长时间保持千兆密封（长达6小时），在5 kHz带宽下实现了低至0.46 pA的RMS噪声值，并且在细胞附着配置中实现了与通过常规膜片钳获得的那些相当的单通道记录。
While high-throughput planar patch-clamp instruments are now established to perform whole-cell recordings for drug screening, the conventional micropipette-based approach remains the gold standard for performing cell-attached single-channel recordings. Generally, planar platforms are not well-suited for such studies due to excess noise resulting from low seal resistances and the use of substrates with poor dielectric properties. Since these platforms tend to use the same pore to position a cell by suction and establish a seal, biological debris from the cell suspension can contaminate the pore surface prior to seal formation, reducing the seal resistance. Here, femtosecond laser ablation was used to fabricate dual-pore glass chips optimized for use in cell-attached single-channel recordings that circumvent this problem by using different pores to position a cell and to establish a seal. This dual-pore design also permitted the use of a relatively small patch aperture (D ~ 150 to 300 nm) that is better-suited for establishing high-resistance seals than the micropores used typically in planar patch-clamp setups (D ~ 1 to 2 μm) without compromising the ability of the device to position a cell. Taking advantage of the high seal resistances and low capacitive and dielectric noise realized using glass substrates, patch-clamp experiments with these dual-pore chips consistently achieved high seal resistances (rate of gigaseal formation = 61%, mean seal resistance = 53 GΩ), maintained gigaseals for prolonged durations (up to 6 hours), achieved RMS noise values as low as 0.46 pA at 5 kHz bandwidth, and enabled single-channel recordings in the cell-attached configuration that are comparable to those obtained by conventional patch-clamp.