Scientists report on an integrated nanopore-based sensing platform that incorporates a microscale preamplifier to improve detection of even the briefest signals emitted by target molecules such as proteins or DNA. The Columbia University Department of Electrical Engineering team claim their technology addresses the limitations of current nanopore-based platforms, which don’t have the temporal resolution to pick up the transient electrical signals generated by small molecules passing through the pores at high speed.
In contrast, reports the Columbia University team and collaborators in Nature Methods, the preamplifier-nanopore platform can pick up signals lasting as little as 1 microsecond that are generated by short DNA molecules whizzing through the sensor pore. “With this platform we achieved a signal-to-noise ratio exceeding five at a bandwidth of 1MHz, which to our knowledge is the highest bandwidth nanopore recording to date,” the investigators state. Their technology and experimental results are detailed in a paper titled “Integrated nanopore sensing platform with sub-microsecond temporal resolution.”
Solid-state nanopore sensors basically comprise an insulating membrane in which a nanometer-scale pore is punched. Both sides of the membrane are exposed to an electrolyte solution, in which an electrode is placed. Applying a voltage to the system then results in a steady-state ion flux through the pore. However, when a single analyte molecule passes through the pore it causes a change in the ionic conductance, and this is measured as an electrical current pulse.
Unfortunately, the fleeting nature of nanopore signals represents a major limitation to this detection approach, the team explains. Typical velocities of nucleic acids passing through solid-state nanopores, for example, are 10–1,000 ns per base, and this may be faster than the measurement time resolution. “Although the signals from nanopores represent a flux of several billion ions per second, in practice nanopore measurements have been constrained to bandwidths below 100 kHz owing to comparatively high background noise,” they write.
Most research focused on improving temporal resolution aims to slow down the DNA as it passes through the nanopore and so increase the duration of signal. In contrast, the Columbia investigators approach is to build faster electronics. In order to achieve this, the team has developed a complementary metal-oxide semiconductor (CMOS)-integrated nanopore platform (CNP), based on a micrometer-scale amplifier optimized for nanopore sensing. The circuitry is positioned directly inside the electrochemical fluid chamber of the sensing device, and the silicon nitride nanopore is placed in the chamber above the amplifier. Their final test design actually contained eight low-noise preamplifiers, and while they used only one channel for subsequent experiments, the investigators stress that the channels are independent and can be operated in parallel.
Using current platforms, pulse durations of 10–100 μs would typically represent the saturation point for temporal resolution, the investigators explain. To test the temporal resolution of their integrated platform, the researchers evaluated its ability to detect the signals generated by 25 bp lengths of double-stranded DNA passing through the nanopore.
They calculated that 29 molecules translocated through the pore in a 500 ms period, producing pulses ranging in duration from just 1.2 μs to 30.2 μs. The sample points were separated by intervals of 0.4 μs, “but the rise and fall times were about 1 μs and about 5 μs for the 500 kHz trace and 100 kHz trace, respectively,” they state. “Accordingly, events shorter than 10 μs were clearly visible in the 500 kHz trace.”
Encouragingly, the CNP sensor experiments were carried out at room temperature, whereas previous attempts to improve the ability of existing nanopore platforms to detect very short binding events have had to be carried out at 0o C to increase the viscosity of the electrolyte and slow the kinetics of surface interactions.
“By introducing a high-performance CMOS preamplifier directly into the electrochemical environment, we extended nanopore signal bandwidths by at least an order of magnitude,” the authors conclude. They believe this improvement will have a multitude of applications in the study of fast reaction kinetics, single-molecule transport, or macromolecular conformation changes. And for potential DNA sequencing applications, the platform will be sensitive enough to support faster translocation and much higher throughput, they add.
Importantly, the platform is highly scalable. “If implemented in a fully dense array, the design presented here would yield 500 preamplifiers per square centimeter, and additional optimizations would enable several thousand independent channels per square centimetre,” the researchers point out. “Paired with appropriate fluidics and an array of biological or solid-state nanopores, this would represent an extraordinarily high-throughput single-molecule sensing platform.”
The team foresees future improvements could boost resolution even further. “With a next-generation design we may be able to get a further 10X improvement and measure things that last only 100 nanoseconds,” the researchers note. “Our lab is also working with other electronic single-molecule techniques based on carbon nanotube transistors, which can leverage similar electronic circuits.”