A new solid‐state, Al2O3 nanopore sensor with enhanced surface properties for the real‐time detection and analysis of individual DNA molecules is reported. Nanopore formation using ...electron‐beam‐based decomposition transforms the local nanostructure and morphology of the pore from an amorphous, stoichiometric structure (O to Al ratio of 1.5) to a heterophase crystalline network, deficient in O (O to Al ratio of ≈0.6). Direct metallization of the pore region is observed during irradiation, thereby permitting the potential fabrication of nanoscale metallic contacts in the pore region with application to nanopore‐based DNA sequencing. Dose‐dependent phase transformations to purely γ and/or α‐phase nanocrystallites are also observed during pore formation, allowing for surface‐charge engineering at the nanopore/fluid interface. DNA transport studies reveal an order‐of‐magnitude reduction in translocation velocities relative to alternate solid‐state architectures, accredited to high surface‐charge density and the nucleation of charged nanocrystalline domains. The unique surface properties of Al2O3 nanopore sensors make them ideal for the detection and analysis of single‐stranded DNA, double‐stranded DNA, RNA secondary structures, and small proteins. These nanoscale sensors may also serve as useful tools in studying the mechanisms driving biological processes including DNA–protein interactions and enzyme activity at the single‐molecule level.
A new solid‐state, Al2O3 nanopore sensor with enhanced surface properties for the real‐time detection and analysis of individual DNA molecules is reported. Nanocrystallite nucleation during pore formation helps enhance the single‐molecule sensitivity and surface‐charge characteristics of these devices and enables the fabrication of nanoscale metal contacts in the pore. This technology finds broad application in drug screening, medicine, and bionanotechnology.
Nanopore analysis is an emerging technique that involves using a voltage to drive molecules through a nanoscale pore in a membrane between two electrolytes, and monitoring how the ionic current ...through the nanopore changes as single molecules pass through it. This approach allows charged polymers (including single-stranded DNA, double-stranded DNA and RNA) to be analysed with subnanometre resolution and without the need for labels or amplification. Recent advances suggest that nanopore-based sensors could be competitive with other third-generation DNA sequencing technologies, and may be able to rapidly and reliably sequence the human genome for under $1,000. In this article we review the use of nanopore technology in DNA sequencing, genetics and medical diagnostics.
Highly sensitive, mechanically robust Al2O3 nanopores are fabricated and characterized. These sensors allow for size control with sub‐nanometer precision, chemical modification, and exhibit superior ...noise performance and increased lifetime over their solid‐state counterparts. This new class of nanopore sensor is used in dsDNA studies and finds broad application in bio‐nanotechnology.
We report the development of a multilayered graphene-Al2O3 nanopore platform for the sensitive detection of DNA and DNA–protein complexes. Graphene-Al2O3 nanolaminate membranes are formed by ...sequentially depositing layers of graphene and Al2O3, with nanopores being formed in these membranes using an electron-beam sculpting process. The resulting nanopores are highly robust, exhibit low electrical noise (significantly lower than nanopores in pure graphene), are highly sensitive to electrolyte pH at low KCl concentrations (attributed to the high buffer capacity of Al2O3), and permit the electrical biasing of the embedded graphene electrode, thereby allowing for three terminal nanopore measurements. In proof-of-principle biomolecule sensing experiments, the folded and unfolded transport of single DNA molecules and RecA-coated DNA complexes could be discerned with high temporal resolution. The process described here also enables nanopore integration with new graphene-based structures, including nanoribbons and nanogaps, for single-molecule DNA sequencing and medical diagnostic applications.
Epigenetic modifications in eukaryotic genomes occur primarily in the form of 5-methylcytosine (5 mC). These modifications are heavily involved in transcriptional repression, gene regulation, ...development and the progression of diseases including cancer. We report a new single-molecule assay for the detection of DNA methylation using solid-state nanopores. Methylation is detected by selectively labeling methylation sites with MBD1 (MBD-1x) proteins, the complex inducing a 3 fold increase in ionic blockage current relative to unmethylated DNA. Furthermore, the discrimination of methylated and unmethylated DNA is demonstrated in the presence of only a single bound protein, thereby giving a resolution of a single methylated CpG dinucleotide. The extent of methylation of a target molecule could also be coarsely quantified using this novel approach. This nanopore-based methylation sensitive assay circumvents the need for bisulfite conversion, fluorescent labeling, and PCR and could therefore prove very useful in studying the role of epigenetics in human disease.
In this paper we present the structure and spectroscopic, photophysical and nonlinear optical studies of covalently functionalized novel graphene oxide-Cu, Zn, Sn, H sub(2) (metal free), VO porphyrin ...composites. The composites were characterized by Field Enhanced Scanning Electron Microscopy (FE-SEM), micro-Raman, optical absorption, Fourier transform infrared (FT-IR), steady state and time resolved fluorescence spectroscopic techniques. The composites exhibit strong fluorescence quenching, suggesting strong electronic interactions between the porphyrin and graphene oxide molecules. Nonlinear optical absorption (NLA) studies of graphene oxide-porphyrin composites were investigated using the Z-scan technique at 532 and 800 nm with nanosecond (ns) and femtosecond (fs) laser pulses. Composites show strong two-photon absorption (TPA) as well as excited state absorption (ESA) leading to reverse saturable absorption (RSA) behaviour in the ns regime and saturable absorption (SA) behaviour was observed in fs regime. The metal free porphyrin-graphene oxide (GO) composite shows significant nonlinear absorption behaviour as well as highest fluorescence quenching behaviour compared to other GO-porphyrin composites. We further observed the enhanced figure of merit (FOM) values for composites in comparison with individual molecules.
Solid-state nanopore sensors are highly versatile platforms for the rapid, label-free electrical detection and analysis of single molecules, applicable to next generation DNA sequencing. The ...versatility of this technology allows for both large scale device integration and interfacing with biological systems. Here we report on the development of a hybrid biological solid-state nanopore platform that incorporates a highly mobile lipid bilayer on a single solid-state Al
2
O
3
nanopore sensor, for the potential reconstitution of ion channels and biological nanopores. Such a system seeks to combine the superior electrical, thermal, and mechanical stability of Al
2
O
3
solid-state nanopores with the chemical specificity of biological nanopores. Bilayers on Al
2
O
3
exhibit higher diffusivity than those formed on TiO
2
and SiO
2
substrates, attributed to the presence of a thick hydration layer on Al
2
O
3
, a key requirement to preserving the biological functionality of reconstituted membrane proteins. Molecular dynamics simulations demonstrate that the electrostatic repulsion between the dipole of the DOPC headgroup and the positively charged Al
2
O
3
surface may be responsible for the enhanced thickness of this hydration layer. Lipid bilayer coated Al
2
O
3
nanopore sensors exhibit excellent electrical properties and enhanced mechanical stability (GΩ seals for over 50 h), making this technology ideal for use in ion channel electrophysiology, the screening of ion channel active drugs and future integration with biological nanopores such as
α
-hemolysin and MspA for rapid single molecule DNA sequencing. This technology can find broad application in bio-nanotechnology.
In this paper, we present a device to electrically count blood cell populations using an AC impedance interrogation technique in a microfabricated cytometer (microcytometer). Specifically, we direct ...our attention to obtaining the concentration of human CD4+ T lymphocytes (helper T cells), which is a necessary method to diagnose patients for HIV/AIDS and to give an accurate prognosis on the effectiveness of ARV (anti-retroviral) drug treatments. We study the effectiveness of a simple-to-fabricate 3-dimensional (3D) hydrodynamic focusing mechanism through fluidic simulations and corresponding experiments to increase the signal-to-noise ratio of impedance pulses caused by particle translocation and ensure lower variance in particle translocation height through the electrical sensing region. We found that the optimal 3D sheath flow settings result in a 44.4% increase in impedance pulse signal-to-noise ratio in addition to giving a more accurate representation of particle size distribution. Our microcytometer T cell counts closely with those found using an industry-standard flow cytometer for the concentration range of over three orders of magnitude and using a sample volume approximately the size of a drop of blood (approximately 20 microL). In addition, our device displayed the capability to differentiate between live and dead/dying lymphocyte populations. This microcytometer can be the basis of a portable, rapid, inexpensive solution to obtaining live/dead blood cell counts even in the most resource-poor regions of the world.
Abstract
A new solid‐state, Al
2
O
3
nanopore sensor with enhanced surface properties for the real‐time detection and analysis of individual DNA molecules is reported. Nanopore formation using ...electron‐beam‐based decomposition transforms the local nanostructure and morphology of the pore from an amorphous, stoichiometric structure (O to Al ratio of 1.5) to a heterophase crystalline network, deficient in O (O to Al ratio of ≈0.6). Direct metallization of the pore region is observed during irradiation, thereby permitting the potential fabrication of nanoscale metallic contacts in the pore region with application to nanopore‐based DNA sequencing. Dose‐dependent phase transformations to purely γ and/or α‐phase nanocrystallites are also observed during pore formation, allowing for surface‐charge engineering at the nanopore/fluid interface. DNA transport studies reveal an order‐of‐magnitude reduction in translocation velocities relative to alternate solid‐state architectures, accredited to high surface‐charge density and the nucleation of charged nanocrystalline domains. The unique surface properties of Al
2
O
3
nanopore sensors make them ideal for the detection and analysis of single‐stranded DNA, double‐stranded DNA, RNA secondary structures, and small proteins. These nanoscale sensors may also serve as useful tools in studying the mechanisms driving biological processes including DNA–protein interactions and enzyme activity at the single‐molecule level.
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