Over the past several decades, a variety of imaging techniques have enabled a wide range of studies of thestructure, function, and dynamics of molecules at the single-molecule level. However, popular fluorescent single-molecule techniques generally cannot directly resolve temporal changes that occur on sub-millisecond timescales, as imaging times must accommodate the relatively slow rate of photon emission from single fluorophores.
In contrast, non-optical techniques that offer direct transduction to ion or electron flux can enable studies of dynamic single-molecule processes on microsecond or nanosecond timescales. Although electronic single-molecule sensors produce larger signals than fluorescent techniques, they are still weak signals, and it is critical to minimize any measurement noise. Towards this end we are designing compact, low-noise, highly parallel, high-speed sensing platforms which combine new direct electronic single-molecule sensors with state-of-the-art semiconductor systems.
For example, a nanopore sensor is a single nanoscale hole in a thin insulating membrane which separates two aqueous solutions. When a target molecule (such as a strand of DNA) passes through a nanopore, it changes the ionic conductance of the pore, which can be measured as an electrical current. Due to the much higher mobility of small dissolved ions compared to larger analyte molecules, nanopores can produce millions of output ions for each individual molecule measured. We recently designed a high-speed nanopore sensing system which combines thin solid-state nanopores with custom low-noise CMOS preamplifiers in a millimeter-scale platform. The low parasitic capacitance of this system allowed us to measure nanopore signals as brief as 1 microsecond, more than 10 times faster than common arrangements based on commercial patch clamp amplifiers.
In addition, we are developing high-speed single-molecule sensors based on chemically functionalized carbon nanotube field-effect transistors. We electrochemically oxidize a carbon nanotube, creating a single point defect which dominates its electronic transport. A probe molecule can be covalently attached to this defect, and the binding of a target molecule to the probe modulates the electron transport through the nanotube. By electrically monitoring the conductance of the nanotube, we can observe single-molecule binding kinetics at very high bandwidth.
Sefi Vernick, Scott M. Trocchia, Steven B. Warren, Erik F. Young, Delphine Bouilly, Ruben L. Gonzalez, Colin Nuckolls & Kenneth L. Shepard. Electrostatic melting in a single-molecule field-effect transistor with applications in genomic identification. Nature Communications 8, Article number: 15450 (2017) | DOI: 10.1038/ncomms15450.
The study of biomolecular interactions at the single-molecule level holds great potential for both basic science and biotechnology applications. Single-molecule studies often rely on fluorescence-based reporting, with signal levels limited by photon emission from single optical reporters. The point-functionalized carbon nanotube transistor, known as the single-molecule field-effect transistor, is a bioelectronics alternative based on intrinsic molecular charge that offers significantly higher signal levels for detection. Such devices are effective for characterizing DNA hybridization kinetics and thermodynamics and enabling emerging applications in genomic identification. In this work, we show that hybridization kinetics can be directly controlled by electrostatic bias applied between the device and the surrounding electrolyte. We perform the first single-molecule experiments demonstrating the use of electrostatics to control molecular binding. Using bias as a proxy for temperature, we demonstrate the feasibility of detecting various concentrations of 20-nt target sequences from the Ebolavirus nucleoprotein gene in a constant-temperature environment
Siddharth Shekar, David J. Niedzwiecki, Chen-Chi Chien, Peijie Ong, Daniel A. Fleischer, Jianxun Lin, Jacob K. Rosenstein, Marija Drndić, Kenneth L. Shepard. Measurement of DNA Translocation Dynamics in a Solid-State Nanopore at 100 ns Temporal Resolution NanoLetters DOI: 10.1021/acs.nanolett.6b01661
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.
J. K. Rosenstein, M. Wanunu, C. A. Merchant, M. Drndic, and K. L. Shepard, “Integrated nanopore sensing platform with sub-microsecond temporal resolution,” doi:10.1038/nmeth.1932, March 18, 2012.
Nanopore sensors have attracted considerable interest for high-throughput sensing of individual nucleic acids and proteins without the need for chemical labels or complex optics. A prevailing problem in nanopore applications is that the transport kinetics of single biomolecules are often faster than the measurement time resolution. Methods to slow down biomolecular transport can be troublesome and are at odds with the natural goal of high-throughput sensing. Here we introduce a low-noise measurement platform that integrates a complementary metal-oxide semiconductor (CMOS) preamplifier with solid-state nanopores in thin silicon nitride membranes. 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. We demonstrate transient signals as brief as 1μs from short DNA molecules as well as current signatures during molecular passage events that shed light on submolecular DNA configurations in small nanopores.
S. Sorgenfrei, C-Y Chiu, R. Gonzalez, Y.-J. Yu, P. Kim, C. Nuckolls, and K. L. Shepard, “Label-free single-molecule detection of DNA hybridization kinetics with a carbon nanotube field-effect transistor,” Nature Nanotechnology 6, pp. 126-132, 2011.
Single-molecule measurements of biomolecules can provide information about the molecular interactions and kinetics that are hidden in ensemble measurements. However, there is a requirement for techniques with improved sensitivity and time resolution for use in exploring biomolecular systems with fast dynamics. Here, we report the detection of DNA hybridization at the single-molecule level using a carbon nanotube field-effect transistor. By covalently attaching a single-stranded probe DNA sequence to a point defect in a carbon nanotube, we are able to measure two-level fluctuations in the conductance of the nanotube in the presence of a complementary DNA target. The kinetics of the system are studied as a function of temperature, allowing the measurement of rate constants, melting curves and activation energies for different sequences and target concentrations. The kinetics demonstrate non-Arrhenius behaviour, in agreement with DNA hybridization experiments using fluorescence correlation spectroscopy. This technique is label-free and could be used to probe single-molecule dynamics at microsecond timescales.