Nanopore-Based Single-Molecule Mass Spectrometry on a Lipid Membrane Microarray

Abstract

We report on parallel high-resolution electrical single-molecule analysis on a chip-based nanopore microarray. Lipid bilayers of <20 μm diameter containing single alpha-hemolysin pores were formed on arrays of subpicoliter cavities containing individual microelectrodes (microelectrode cavity array, MECA), and ion conductance-based single molecule mass spectrometry was performed on mixtures of poly(ethylene glycol) molecules of different length. We thereby demonstrate the function of the MECA device as a chip-based platform for array-format nanopore recordings with a resolution at least equal to that of established single microbilayer supports. We conclude that devices based on MECAs may enable more widespread analytical use of nanopores by providing the high throughput and ease of operation of a high-density array format while maintaining or exceeding the precision of state-of-the-art microbilayer recordings.

Functional Analysis of Membrane Proteins Produced by Cell-Free Translation

“…After incubating the lipid bilayer with the proteoliposomes, measure activity from the voltage-clamped lipid bilayers of the sec61 pore by a single channel amplifier single channel amplifier (EPC-10) and the data acquisition software Patchmaster connected to the multiplexer electronics port of the Orbit16 system [14][15][16]. Recordings are done at a sampling rate of 50 kHz with a 10 kHz Bessel filter (see Subheading 2.6, step 7).…”

Scalable micro-cavity bilayer lipid membrane arrays for parallel ion channel recording

“…In order to increase throughput, bilayer arrays have been developed, for example using stereo-lithography to define chambers around a thin film of a hydrophobic polymer that includes an aperture for bilayer formation [9,10]. Both ease of bilayer formation and stability has been improved through the use of small cavities in the range 6-50 m diameter [11][12][13]. However, small cavities have small in situ electrodes, which limits the chip lifetime or measurement duration [11,14].…”

“…Both ease of bilayer formation and stability has been improved through the use of small cavities in the range 6-50 m diameter [11][12][13]. However, small cavities have small in situ electrodes, which limits the chip lifetime or measurement duration [11,14]. Baaken et al developed a 4 × 4 array of micro-cavities using a single layer of SU8 [11], and Ogier et al fabricated micron sized cavities using a combination of SU8 and polymer films [12].…”

“…However, small cavities have small in situ electrodes, which limits the chip lifetime or measurement duration [11,14]. Baaken et al developed a 4 × 4 array of micro-cavities using a single layer of SU8 [11], and Ogier et al fabricated micron sized cavities using a combination of SU8 and polymer films [12]. Suzuki et al fabricated a 96 well planar bilayer structure using stereolithography, with apertures patterned in parylene [13].…”

“…Devices have also been made by bonding parylene film with pre-formed apertures to a backplane made using stereolithography [13]; parallel electrical recording was not possible and each well was sequentially switched to a recording amplifier [13]. Parallel recording using large commercial multi-channel (8 channel) amplifiers has been reported [9,11,16]. This approach is costly and the long wires increase electrical noise.…”