Abstract
Diffracted X-ray tracking (DXT) method can trace conformational changes of KcsA potassium ion channel during gating by recording position of diffraction spot from a gold nanocrystal attached to the channel as a movie. For high-resolution imaging under controlled microenvironments for KcsA channels, we report a microfluidic device consisting of two SiN membrane windows bonded with a photo patternable adhesive material. The reduced signal-to-background ratio as well as suitable adhesive material thickness for the microchannel are discussed in the experiment at the synchrotron radiation facility.
Highlights
Activity of ion channel is modulated by relevant signals that tightly control the opening and closing of the channel
We propose a new experimental setup for the diffracted X-ray tracking (DXT) method that control microenvironments for KcsA channels fixed in an observation chamber, leading to capture conformational change occurring in response to chemicals
In order to determine a suitable microchannel height, background noise generated by observation chamber was evaluated
Summary
Activity of ion channel is modulated by relevant signals (e.g., chemical or optical) that tightly control the opening and closing of the channel. We propose a new experimental setup for the DXT method that control microenvironments for KcsA channels fixed in an observation chamber, leading to capture conformational change occurring in response to chemicals (e.g., acidic pH or ligands) This observation setup present here differs from the previous work [4] in the specific fabrication for improving signal-to-background (S/B) ratio by assembling SiN membrane windows. KcsA channels were conventionally fixed on a glass substrate having a thickness of 50 μm, and they were covered by thin polyimide film for constructing the observation chamber This observation setup successfully captured the conformational changes of single KcsA channels, high background noise due to X-ray scattering of the observation chamber has limited the record of the diffraction spots, and spatial resolution was not good enough (Figure 1b). A fabrication of new microfluidic structure in Figure 2b stars with, (1) SiN was patterned by UV lithography for subsequent KOH etching (2), which releases a SiN membrane having a thickness of
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