Wavelet Denoising of High-Bandwidth Nanopore and Ion-Channel Signals.

Recent work has pushed the noise-limited bandwidths of solid-state nanopore conductance recordings to more than 5 MHz and of ion channel conductance recordings to more than 500 kHz through the use of integrated complementary metal-oxide-semiconductor (CMOS) integrated circuits. Despite the spectral spread of the pulse-like signals that characterize these recordings when a sinusoidal basis is employed, Bessel filters are commonly used to denoise these signals to acceptable signal-to-noise ratios (SNRs) at the cost of losing many of the faster temporal features. Here, we report improvements to the SNR that can be achieved using wavelet denoising instead of Bessel filtering. When combined with state-of-the-art high-bandwidth CMOS recording instrumentation, we can reduce baseline noise levels by over a factor of 4 compared to a 2.5 MHz Bessel filter while retaining transient properties in the signal comparable to this filter bandwidth. Similarly, for ion-channel recordings, we achieve a temporal response better than a 100 kHz Bessel filter with a noise level comparable to that achievable with a 25 kHz Bessel filter. Improvements in SNR can be used to achieve robust statistical analyses of these recordings, which may provide important insights into nanopore translocation dynamics and mechanisms of ion-channel function.

Single-channel recordings of RyR1 at microsecond resolution in CMOS-suspended membranes.

Single-channel recordings are widely used to explore functional properties of ion channels. Typically, such recordings are performed at bandwidths of less than 10 kHz because of signal-to-noise considerations, limiting the temporal resolution available for studying fast gating dynamics to greater than 100 µs. Here we present experimental methods that directly integrate suspended lipid bilayers with high-bandwidth, low-noise transimpedance amplifiers based on complementary metal-oxide-semiconductor (CMOS) integrated circuits (IC) technology to achieve bandwidths in excess of 500 kHz and microsecond temporal resolution. We use this CMOS-integrated bilayer system to study the type 1 ryanodine receptor (RyR1), a Ca2+-activated intracellular Ca2+-release channel located on the sarcoplasmic reticulum. We are able to distinguish multiple closed states not evident with lower bandwidth recordings, suggesting the presence of an additional Ca2+ binding site, distinct from the site responsible for activation. An extended beta distribution analysis of our high-bandwidth data can be used to infer closed state flicker events as fast as 35 ns. These events are in the range of single-file ion translocations.

Measurement of DNA Translocation Dynamics in a Solid-State Nanopore at 100 ns Temporal Resolution.

Despite the potential for nanopores to be a platform for high-bandwidth study of single-molecule systems, ionic current measurements through nanopores have been limited in their temporal resolution by noise arising from poorly optimized measurement electronics and large parasitic capacitances in the nanopore membranes. Here, we present a complementary metal-oxide-semiconductor (CMOS) nanopore (CNP) amplifier capable of low noise recordings at an unprecedented 10 MHz bandwidth. When integrated with state-of-the-art solid-state nanopores in silicon nitride membranes, we achieve an SNR of greater than 10 for ssDNA translocations at a measurement bandwidth of 5 MHz, which represents the fastest ion current recordings through nanopores reported to date. We observe transient features in ssDNA translocation events that are as short as 200 ns, which are hidden even at bandwidths as high as 1 MHz. These features offer further insights into the translocation kinetics of molecules entering and exiting the pore. This platform highlights the advantages of high-bandwidth translocation measurements made possible by integrating nanopores and custom-designed electronics.