Graphene: A sub-nanometer trans-electrode membrane

Isolated, atomically thin conducting membranes of graphite, called graphene, have recently been the subject of intense research with the hope that practical applications in fields ranging from electronics to energy science will emerge. Here, we show that when immersed in ionic solution, a layer of graphene takes on new electrochemical properties that make it a trans-electrode. The trans-electrode's properties are the consequence of the atomic scale proximity of its two opposing liquid-solid interfaces together with graphene's well known in-plane conductivity. We show that several trans-electrode properties are revealed by ionic conductivity measurements on a CVD grown graphene membrane that separates two aqueous ionic solutions. Despite this membrane being only one to two atomic layers thick, we find it is a remarkable ionic insulator with a very small stable conductivity that depends on the ion species in solution. Electrical measurements on graphene membranes in which a single nanopore has been drilled show that the membrane's effective insulating thickness is less than one nanometer. This small effective thickness makes graphene an ideal substrate for very high-resolution, high throughput nanopore based single molecule detectors. Sensors based on modulation of graphene's in-plane electronic conductivity in response to trans-electrode environments and voltage biases will provide new insights into atomic processes at the electrode surfaces.Comment: Submitted 12 April 2010 to Nature, where it is under revie

Nucleotide Discrimination with DNA Immobilized in the MspA Nanopore

Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices

Single-molecule sensing electrode embedded in-plane nanopore

Electrode-embedded nanopore is considered as a promising device structure for label-free single-molecule sequencing, the principle of which is based on nucleotide identification via transverse electron tunnelling current flowing through a DNA translocating through the pore. Yet, fabrication of a molecular-scale electrode-nanopore detector has been a formidable task that requires atomic-level alignment of a few nanometer sized pore and an electrode gap. Here, we report single-molecule detection using a nucleotide-sized sensing electrode embedded in-plane nanopore. We developed a self-alignment technique to form a nanopore-nanoelectrode solid-state device consisting of a sub-nanometer scale electrode gap in a 15 nm-sized SiO2 pore. We demonstrate single-molecule counting of nucleotide-sized metal-encapsulated fullerenes in a liquid using the electrode-integrated nanopore sensor. We also performed electrical identification of nucleobases in a DNA oligomer, thereby suggesting the potential use of this synthetic electrode-in-nanopore as a platform for electrical DNA sequencing

Transverse electric field dragging of DNA in a nanochannel

Nanopore analysis is an emerging single-molecule strategy for non-optical and high-throughput DNA sequencing, the principle of which is based on identification of each constituent nucleobase by measuring trans-membrane ionic current blockade or transverse tunnelling current as it moves through the pore. A crucial issue for nanopore sequencing is the fact that DNA translocates a nanopore too fast for addressing sequence with a single base resolution. Here we report that a transverse electric field can be used to slow down the translocation. We find 400-fold decrease in the DNA translocation speed by adding a transverse field of 10 mV/nm in a gold-electrode-embedded silicon dioxide channel. The retarded flow allowed us to map the local folding pattern in individual DNA from trans-pore ionic current profiles. This field dragging approach may provide a new way to control the polynucleotide translocation kinetics

Support Vector Machine Implementations for Classification & Clustering

BACKGROUND: We describe Support Vector Machine (SVM) applications to classification and clustering of channel current data. SVMs are variational-calculus based methods that are constrained to have structural risk minimization (SRM), i.e., they provide noise tolerant solutions for pattern recognition. The SVM approach encapsulates a significant amount of model-fitting information in the choice of its kernel. In work thus far, novel, information-theoretic, kernels have been successfully employed for notably better performance over standard kernels. Currently there are two approaches for implementing multiclass SVMs. One is called external multi-class that arranges several binary classifiers as a decision tree such that they perform a single-class decision making function, with each leaf corresponding to a unique class. The second approach, namely internal-multiclass, involves solving a single optimization problem corresponding to the entire data set (with multiple hyperplanes). RESULTS: Each SVM approach encapsulates a significant amount of model-fitting information in its choice of kernel. In work thus far, novel, information-theoretic, kernels were successfully employed for notably better performance over standard kernels. Two SVM approaches to multiclass discrimination are described: (1) internal multiclass (with a single optimization), and (2) external multiclass (using an optimized decision tree). We describe benefits of the internal-SVM approach, along with further refinements to the internal-multiclass SVM algorithms that offer significant improvement in training time without sacrificing accuracy. In situations where the data isn't clearly separable, making for poor discrimination, signal clustering is used to provide robust and useful information – to this end, novel, SVM-based clustering methods are also described. As with the classification, there are Internal and External SVM Clustering algorithms, both of which are briefly described

A novel, fast, HMM-with-Duration implementation – for application with a new, pattern recognition informed, nanopore detector

<p>Abstract</p> <p>Background</p> <p>Hidden Markov Models (HMMs) provide an excellent means for structure identification and feature extraction on stochastic sequential data. An HMM-with-Duration (HMMwD) is an HMM that can also exactly model the hidden-label length (recurrence) distributions – while the regular HMM will impose a best-fit geometric distribution in its modeling/representation.</p> <p>Results</p> <p>A Novel, Fast, HMM-with-Duration (HMMwD) Implementation is presented, and experimental results are shown that demonstrate its performance on two-state synthetic data designed to model Nanopore Detector Data. The HMMwD experimental results are compared to (i) the ideal model and to (ii) the conventional HMM. Its accuracy is clearly an improvement over the standard HMM, and matches that of the ideal solution in many cases where the standard HMM does not. Computationally, the new HMMwD has all the speed advantages of the conventional (simpler) HMM implementation. In preliminary work shown here, HMM feature extraction is then used to establish the first pattern recognition-informed (PRI) sampling control of a Nanopore Detector Device (on a "live" data-stream).</p> <p>Conclusion</p> <p>The improved accuracy of the new HMMwD implementation, at the same order of computational cost as the standard HMM, is an important augmentation for applications in gene structure identification and channel current analysis, especially PRI sampling control, for example, where speed is essential. The PRI experiment was designed to inherit the high accuracy of the well characterized and distinctive blockades of the DNA hairpin molecules used as controls (or blockade "test-probes"). For this test set, the accuracy inherited is 99.9%.</p

Nanopore Detector based analysis of single-molecule conformational kinetics and binding interactions

BACKGROUND: A Nanopore Detector provides a means to transduce single molecule events into observable channel current changes. Nanopore-based detection can report directly, or indirectly, on single molecule kinetics. The nanopore-based detector can directly measure molecular characteristics in terms of the blockade properties of individual molecules – this is possible due to the kinetic information that is embedded in the blockade measurements, where the adsorption-desorption history of the molecule to the surrounding channel, and the configurational changes in the molecule itself, imprint on the ionic flow through the channel. This rich source of information offers prospects for DNA sequencing and single nucleotide polymorphism (SNP) analysis. A nanopore-based detector can also measure molecular characteristics indirectly, by using a reporter molecule that binds to certain molecules, with subsequent distinctive blockade by the bound-molecule complex. RESULTS: It is hypothesized that reaction histories of individual molecules can be observed on model DNA/DNA, DNA/Protein, and Protein/Protein systems. Preliminary results are all consistent with this hypothesis. Nanopore detection capabilities are also described for highly discriminatory biosensing, binding strength characterization, and rapid immunological screening. CONCLUSION: In essence, the heart of chemistry is now accessible to a new, single-molecule, observation method that can track both external molecular binding states, and internal conformation states