Bala Murali Venkatesan

Nanopore sensors for nucleic acid analysis

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

Stacked Graphene-Al2O3 Nanopore Sensors for Sensitive Detection of DNA and DNA–Protein Complexes

1,000. In this article we review the use of nanopore technology in DNA sequencing, genetics and medical diagnostics.

Detection and Quantification of Methylation in DNA using Solid-State Nanopores

We report the development of a multilayered graphene-Al(2)O(3) nanopore platform for the sensitive detection of DNA and DNA-protein complexes. Graphene-Al(2)O(3) nanolaminate membranes are formed by sequentially depositing layers of graphene and Al(2)O(3), 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 Al(2)O(3)), 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.

A robust electrical microcytometer with 3-dimensional hydrofocusing

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.

Solid-State Nanopore Sensors for Nucleic Acid Analysis

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.

Nanopore sensors for DNA analysis

Solid-state nanopores are nm sized apertures formed in thin synthetic membranes. These single molecule sensors have been used in a variety of biophysical and diagnostic applications and serve as a potential candidate in the development of cost-effective, next generation DNA sequencing technologies, critical to furthering our understanding of inheritance, individuality, disease and evolution. The versatility of solid-state nanopore technology allows for both interfacing with biological systems at the nano-scale as well as large scale VLSI integration promising reliable, affordable, mass producible biosensors with single molecule sensing capabilities. In addition, this technology allows for truly parallel, high throughput DNA and protein analysis through the development of nanopore and micropore arrays in ultra-thin synthetic membranes. This chapter is focused on the development of solid-state nanopore sensors in synthetic membranes and the potential benefits and challenges associated with this technology. Biological nanopores, primarily α-hemolysin and the phi29 connector are also reviewed. We conclude with a detailed discussion on chemically modified solid-state nanopores. These surface functionalized nanopore sensors combine the stability and versatility of solid-state nanopores with the sensitivity and selectivity of biological nanopore systems and may play an important role in drug screening and medical diagnostics.

Al 2 O 3 Nanopore Sensors for Single Molecule DNA Detection

Solid-state nanopore sensors are promising devices for single DNA molecule detection and sequencing. This paper presents a review of our work on solid-state nanopores performed over the last decade. In particular, here we discuss atomic-layer-deposited (ALD)-based, graphene-based, and functionalized solid state nanopores.

Highly Sensitive, Mechanically Stable Nanopore Sensors for DNA Analysis

We report the development of a new solid-state, Al2O3 nanopore sensor with enhanced surface properties for the real-time detection and analysis of individual DNA molecules. The nanopore fabrication process involves first forming, free-standing, 45 ± 5 nm thick Al2O3 membranes using standard micro-fabrication techniques. Next, a focused convergent electron beam from a field emission Transmission Electron Microscope (TEM) is used to decompositionally sputter single nanopores in these ultra-thin Al2O3 membranes. These nanopores exhibit excellent mechanical properties (high mechanical hardness, low stress) and state-of-the-art electrical performance (low noise, high signal-to-noise ratio), making them ideal for single molecule DNA analysis. Furthermore, we report drastic changes in the material properties of the nanopore during pore formation. Prolonged electron beam irradiation induces changes in the local stoichiometry and morphology of the pore from an amorphous, stoichiometric structure (O to Al ratio of 1.5) to a hetero-phase, crystalline structure with a nonstoichiometric O to Al ratio of ~0.6. Preferential phase transformations from γ, α, κ and δ-Al2O3 to purely γ and α-phases are observed with increasing electron dose. Precise control over phase transformations in Al2O3 nanopore systems may provide a novel method to engineer surface charge at the nanopore/fluid interface. Metallization of the irradiated region is also observed, attributed to the preferential desorption of O and the aggregation of metallic Al clusters as confirmed through nanoarea electron diffraction and electron energy loss spectroscopy in the TEM. This in-situ metallization process may be applicable to the fabrication of nano-scale metallic contacts directly in the nanopore thereby enabling the electrical manipulation of surface charge and nanopore conductance.