Search

Nanopores based on protein channels inserted into lipid membranes have paved the way towards a wide-range of inexpensive biosensors, especially for DNA sequencing. A key obstacle in using these biological ion channels as nanodevices is the poor stability of lipid bilayer membranes. Amphiphilic block copolymer membranes have emerged as a robust alternative to lipid membranes. While previous efforts have shown feasibility, we demonstrate for the first time the effect of polymer composition on MspA protein functionality. We show that membrane-protein interaction depends on the hydrophobic-hydrophilic ratio (f-ratio) of the block copolymer. These effects are particularly pronounced in asymmetric protein pores like MspA compared to the cylindrical α-Hemolysin pore. A key effect of membrane-protein interaction is the increased 1/fα noise. After first showing increases in 1/fα behaviour arise from increased substate activity, the noise power spectral density S(f) was used as a qualitative tool for understanding protein-membrane interactions in polymer membranes. Polymer compositions with f-ratios close to lipid membranes caused noise behaviour not observed in lipid membranes. However, by modifying the f-ratio using a modular synthetic approach, we were able to design a block copolymer exhibiting noise properties similar to a lipid membrane, albeit with better stability. Thus, by careful optimization, block copolymer membranes can emerge as a robust alternative for protein-pore based nano-biosensors.

Voltage gating is essential to the computational ability of neurons. We show this effect can be mimicked in a solid-state nanopore by functionalizing the pore interior with a redox active molecule. We study the integration of an active biological molecule-a quinone-into a solid state nanopore, and its subsequent induced voltage gating. We show that the voltage gating effect mimics biological gating systems in its classic sigmoidal voltage response, unlike previous synthetic voltage gating systems. Initially, the quinone undergoes a reduction due to radicals in the bulk solution, and is converted to the hydroquinone state. Upon deprontonation the hydroquinone then acts as a charged nanomechanical arm, which opens the channel under the applied potential. We establish that the quinone gains a single net charge when the pH inside of the nanopore reaches its pKa value, and explore factors that influence the net pH in the middle of the pore. Using a combination of theory, experiment and simulation, we conclude that concentration polarization and a shift of the pH inside of the channel is the main cause of this gating effect.

Understanding the biophysics governing single-molecule transport through solid-state nanopores is of fundamental importance in working toward the goal of DNA detection and genome sequencing using nanopore-based sensors. Even with significant advances in semiconductor fabrication technologies, the state-of-the-art in nanopore technology still falls well short of mimicking the elegance and functionality found in biology. Kasianowicz et al.[1] pioneered the first in vitro studies of biomolecule transport through single nanopore channels by translocating individual ssDNA and ssRNA molecules through α-hemolysin protein pores inserted into a lipid bilayer membrane. More recently, focus has shifted to the solid-state domain with numerous groups studying biomolecule transport through solid-state nanopores.[2-7] Solid-state nanopores exhibit superior chemical, thermal, and mechanical stability over their biological counterparts, and can be fabricated using conventional semiconductor processes, thereby facilitating mass fabrication and size tunability. They are typically formed in thin Si3N4 or SiO2 membranes using a combination of decompositional ion/electron-beam-based sputtering and surface-tension-driven shrinking processes.[2,4,7] Other techniques for creating individual nanopores include the track-etch method for the formation of conical nanopores in polycarbonate membranes.[8] The translocation of negatively charged DNA molecules through these nanometer-sized solid-state pores is conventionally performed using two-terminal electrophoresis, resulting in characteristic blockades in the measured ionic current. This technique has been used to study various physical phenomena at the single-molecule level, including unzipping kinetics of hairpin DNA,[9] detection of single-nucleotide polymorphisms,[10] stretching transitions in dsDNA,[11] biomolecule folding,[3] discrimination of long DNA molecules based on length,[12] and nanopore-based DNA force spectroscopy.[13] Though this technology shows much promise, major hurdles still remain. Fabrication challenges (stress-induced membrane deformation and mechanical failure in SiO2 structures),[5] limited nanopore lifetime, electrical noise,[14,15] and a lack of chemical specificity limit the feasibility of this technology in high-end applications such as DNA sequencing. Thus, there is a need for highly sensitive, mechanically robust nanopore sensors with well-defined surface-charge properties for the detection of specific biological molecules (ssDNA, dsDNA, mRNA).

We have explored the electromechanical properties of DNA by using an electric field to force single hairpin molecules to translocate through a synthetic pore in a silicon nitride membrane. We observe a threshold voltage for translocation of the hairpin through the pore that depends sensitively on the diameter and the secondary structure of the DNA. The threshold for a diameter 1.5 < d < 2.3 nm is V > 1.5 V, which corresponds to the force required to stretch the stem of the hairpin, according to molecular dynamics simulations. On the other hand, for 1.0 < d < 1.5 nm, the threshold voltage collapses to V < 0.5 V because the stem unzips with a lower force than required for stretching. The data indicate that a synthetic nanopore can be used like a molecular gate to discriminate between the secondary structures in DNA.

Solid-state nanopores have emerged as a single molecule label-free electronic detection platform. Existing transimpedance stages used to measure ionic current nanopores suffer from dynamic range limitations resulting from steady-state baseline currents. We propose a digitally-assisted baseline cancellation CMOS platform that circumvents this issue. Since baseline cancellation is a form of auto-zeroing, the 1/f noise of the system is also reduced. Our proposed design can tolerate a steady state baseline current of 10μA and has a usable bandwidth of 750kHz. Quantitative DNA translocation experiments on 5kbp DNA was performed using a 5nm silicon nitride pore using both the CMOS platform and a commercial system. Comparison of event-count histograms show that the CMOS platform clearly outperforms the commercial system, allowing for unambiguous interpretation of the data.

This paper outlines our recent eorts in using solid-state nanopores as a biosensing platform. Traditionally biosensors concentrate mainly on the detection platform and not on signal processing. This decoupling can lead to inferior sensors and is exacerbated in nanoscale devices, where device noise is large and large dynamic range is required. This paper outlines a novel platform that integrates the nano, micro and macroscales in a closely coupled manner that mitigates many of these problems. We discuss our initial results of DNA translocation through the nanopore. We also briey discuss the use of molecular recognition properties of aptamers with the versatility of the nanopore detector to design a new class of biosensors in a CMOS compatible platform.

A nanopore is an analytical tool with single molecule sensitivity. For detection, a nanopore relies on the electrical signal that develops when a molecule translocates through it. However, the detection sensitivity can be adversely affected by noise and the frequency response. Here, we report measurements of the frequency and noise performance of nanopores

High-bandwidth and low-noise nanopore sensor and detection electronics are crucial in achieving single-DNA-base resolution. A potential way to accomplish this goal is to integrate solid-state nanopores within a CMOS platform, in close proximity to the biasing electrodes and custom-designed amplifier electronics. Here we report the integration of solid-state nanopore devices in a commercial complementary metal-oxide-semiconductor (CMOS) potentiostat chip implemented in On-Semiconductor's 0.5 μm technology. Nanopore membranes incorporating electrodes are fabricated by post-CMOS micromachining utilizing the n+ polysilicon/SiO2/n+ polysilicon capacitor structure available in the aforementioned process. Nanopores are created in the CMOS process by drilling in a transmission electron microscope and shrinking by atomic layer deposition. We also describe a batch fabrication method to process a large of number of electrode-embedded nanopores with sub-10 nm diameter across CMOS-compatible wafers by electron beam lithography and atomic layer deposition. The CMOS-compatibility of our fabrication process is verified by testing the electrical functionality of on-chip circuitry. We observe high current leakage with the CMOS nanopore devices due to the ionic diffusion through the SiO2 membrane. To prevent this leakage, we coat the membrane with Al2O3, which acts as an efficient diffusion barrier against alkali ions. The resulting nanopore devices also exhibit higher robustness and lower 1/f noise as compared to SiO2 and SiNx. Furthermore, we propose a theoretical model for our low-capacitance CMOS nanopore devices, showing good agreement with the experimental value. In addition, experiments and theoretical models of translocation studies are presented using 48.5 kbp λ-DNA in order to prove the functionality of on-chip pores coated with Al2O3.

Understanding the biophysics governing single molecule transport through solid state nanopores is of fundamental importance in working towards the goal of genome sequencing using nanopore based sensors. Here, we present a simple process for the fabrication and characterization of novel, low stress, low noise aluminum oxide nanopores for biomolecule detection. Aluminum oxide has numerous attractive properties including high mechanical hardness, low surface charge, chemical inertness to strong acids and excellent dielectric properties from DC to GHz frequencies.Device fabrication involved the use of Atomic Layer Deposition and Deep Reactive Ion Etching tools to form low stress, mechanically robust aluminum oxide membranes. High temperature process steps were avoided to allow for possible process integration with metal nano-electrodes and optical probes. The nanometer sized pores themselves were formed through Field Emission Gun Transmission Electron Microscope (FEG-TEM) based sputtering. We demonstrate the precise size tunability of these structures in the nanometer regime and examine the physics governing pore contraction in aluminum oxide. Diffraction patterns reveal polycrystallinity localized to the pore region post sputtering suggesting localized heating and possible thermal annealing under the electron beam. Film composition and thickness were characterized. In addition, we examine the surface charge properties of these structures as a function of buffer pH and molarity. The single molecule sensing ability of this novel structure was tested using dsDNA. Electrical characterization revealed a significant reduction in membrane capacitance and reduced high frequency dielectric noise relative to existing silicon nitride and silicon dioxide topologies. These improvements can greatly enhance device performance by improving sensitivity and signal-to-noise ratio. In summary, our work provides a novel yet simple approach to fabricate tunable, low stress chemically functionalizable nanopores for the detection of biomolecules.