Andre Marziali

Noise analysis and reduction in solid-state nanopores

The electrical noise characteristics of ionic current through organic and synthetic nanopores have been investigated. Comparison to proteinaceous alpha-Hemolysin pores reveals two dominant noise sources in silicon nitride nanometre-scale pores: a high-frequency noise associated with the capacitance of the silicon support chip (dielectric noise), and a low-frequency current fluctuation with 1/ f α characteristics (flicker noise). We present a technique for reducing the dielectric noise by curing polydimethylsiloxane (PDMS) on the nanopore support chip. This greatly improves the performance of solid-state nanopore devices, yielding an unprecedented signal-to-noise ratio when observing dsDNA translocation events and ssDNA probe capture for force spectroscopy applications. (Some figures in this article are in colour only in the electronic version)

Nanopore sensors for nucleic acid analysis

In the past decade, nanometre-scale pores have been explored as the basis for technologies to analyse and sequence single nucleic acid molecules. Most approaches involve using such a pore to localize single macromolecules and interact with them to garner some information on their composition. Though nanopore sensors cannot yet claim success at deoxyribonucleic acid (DNA) sequencing, nanopore-based technologies offer one of the most promising approaches to single molecule detection and analysis. The majority of experimental work with nanopore detection of nucleic acids has involved the α-haemolysin (alpha-HL) ion channel—a heptameric protein with a ~2 nm diameter inner pore which allows translocation of single-stranded DNA. Analysis of externally induced ion current through the pore during its interaction with DNA can provide information about the DNA molecule, including length and base composition. This review focuses on alpha-HL and its applications to single-molecule detection. Modified alpha-HL and other biological and synthetic pores for macromolecule detection are also discussed, along with a brief summary of relevant theoretical work and numerical modelling of polymer–pore interaction.

Evaluation of nanopores as candidates for electronic analyte detection.

In an effort to increase throughput and decrease the cost of electrophoretic separation of DNA and proteins, various groups are developing highly parallel, miniaturized separation devices based on capillaries etched into silicon, glass or plastic substrates. To date, these miniaturized devices have relied on optical detectors, thus placing a lower limit on instrument size, and complicating the incorporation of an entire DNA analyzer instrument on a chip. To address this limitation, we are evaluating nanopores as candidate Coulter counters for purely electronic detection of analytes in miniaturized electrophoresis and similar separation devices. To establish feasibility of this detection scheme, we have investigated the detection sensitivity of a nanopore sensor through experiments with the α‐hemolysin (α‐HL) ion channel, and through a Monte Carlo (MC) model of polymer capture rate with a cylindrical nanopore under an applied voltage. Experimental and model results are extrapolated to predict the capture rate of synthetic pores operating at higher voltages than presently achievable with protein pores.

Single-molecule bonds characterized by solid-state nanopore force spectroscopy.

Weak molecular interactions drive processes at the core of living systems, such as enzyme-substrate interactions, receptor-ligand binding, and nucleic acid replication. Single-molecule force spectroscopy is a remarkable tool for revealing molecular scale energy landscapes of noncovalent bonds, by exerting a mechanical force directly on an individual molecular complex and tracking its survival as a function of time and applied force. In principle, force spectroscopy methods can also be used for highly specific molecular recognition assays, by directly characterizing the strength of bonds between probe and target molecules. However, complexity and low throughput of conventional force spectroscopy techniques render such biosensing applications impractical. Here we demonstrate a straightforward single-molecule approach, suitable for both biophysical studies and molecular recognition assays, in which a approximately 3 nm silicon nitride nanopore is used to determine the bond lifetime spectrum of the biotin-neutravidin complex. Thousands of individual molecular complexes are captured and dissociated in the solid-state nanopore under constant applied forces, ranging from 400 to 900 mV, allowing us to extract the location of the energy barrier that governs the interaction, mapped at Deltax approximately 0.5 nm. These results highlight the capacity of a solid-state nanopore to detect and characterize intermolecular interactions and demonstrate how this could be applied to rapid, highly specific molecular detection assays.

Nonlinear electrophoretic response yields a unique parameter for separation of biomolecules

We demonstrate a unique parameter for biomolecule separation that results from the nonlinear response of long, charged polymers to electrophoretic fields and apply it to extraction and concentration of nucleic acids from samples that perform poorly under conventional methods. Our method is based on superposition of synchronous, time-varying electrophoretic fields, which can generate net drift of charged molecules even when the time-averaged molecule displacement generated by each field individually is zero. Such drift can only occur for molecules, such as DNA, whose motive response to electrophoretic fields is nonlinear. Consequently, we are able to concentrate DNA while rejecting high concentrations of contaminants. We demonstrate one application of this method by extracting DNA from challenging samples originating in the Athabasca oil sands.

An instrument for automated purification of nucleic acids from contaminated forensic samples

Forensic crime scene sample analysis, by its nature, often deals with samples in which there are low amounts of nucleic acids, on substrates that often lead to inhibition of subsequent enzymatic reactions such as PCR amplification for Short Tandem Repeat (STR) profiling. Common substrates include denim from blue jeans, which yields indigo dye as a PCR inhibitor, and soil, which yields humic substances as inhibitors. These inhibitors frequently co-extract with nucleic acids in standard column or bead-based preps, leading to frequent failure of STR profiling. We present a novel instrument for DNA purification of forensic samples that is capable of highly effective concentration of nucleic acids from soil particulates, fabric, and other complex samples including solid components. The novel concentration process, known as Synchronous Coefficient of Drag Alteration, is inherently selective for long-charged polymers such as DNA, and therefore is able to effectively reject known contaminants. We present an automated sample preparation instrument based on this process, and preliminary results based on mock forensic samples.

Nonexponential Kinetics of DNA Escape from α-Hemolysin Nanopores ☆

Throughput and resolution of DNA sequence detection technologies employing nanometer scale pores hinge on accurate kinetic descriptions of DNA motion in nanopores. We present the first detailed experimental study of DNA escape kinetics from alpha-hemolysin nanopores and show that anomalously long escape times for some events result in nonexponential kinetics. From the distribution of first-passage times, we determine that the energy barrier to escape follows a Poisson-like distribution, most likely due to stochastic weak binding events between the DNA and amino acid residues in the pore.

Evidence that small proteins translocate through silicon nitride pores in a folded conformation

The interaction of three proteins (histidine-containing phosphocarrier protein, HPr, calmodulin, CaM, and maltose binding protein, MBP) with synthetic silicon nitride (SiN(x)) membranes has been studied. The proteins which have a net negative charge were electrophoretically driven into pores of 7 and 5 nm diameter with a nominal length of 15 nm. The % blockade current and event duration were measured at three different voltages. For a translocation event it was expected that the % block would be constant with voltage whilst the event duration would decrease with increasing voltage. On the basis of these criteria, we deduce that MBP whose largest dimension is 6.5 nm does not translocate whereas up to 40% of CaM molecules can translocate the 7 nm pore as can a majority of HPr molecules, with some translocations being observed for the 5 nm pore. For translocation events the magnitude of the % blockade current is consistent with a folded conformation of the proteins surrounded by a hydration shell of 0.5-1.0 nm.

Portable dynamic light scattering instrument and method for the measurement of blood platelet suspensions

No routine test exists to determine the quality of blood platelet transfusions although every year millions of patients require platelet transfusions to survive cancer chemotherapy, surgery or trauma. A new, portable dynamic light scattering instrument is described that is suitable for the measurement of turbid solutions of large particles under temperature-controlled conditions. The challenges of small sample size, short light path through the sample and accurate temperature control have been solved with a specially designed temperature-controlled sample holder for small diameter, disposable capillaries. Efficient heating and cooling is achieved with Peltier elements in direct contact with the sample capillary. Focusing optical fibres are used for light delivery and collection of scattered light. The practical use of this new technique was shown by the reproducible measurement of latex microspheres and the temperature-induced morphological changes of human blood platelets. The measured parameters for platelet transfusions are platelet size, number of platelet-derived microparticles and the response of platelets to temperature changes. This three-dimensional analysis provides a high degree of confidence for the determination of platelet quality. The experimental data are compared to a matrix and facilitate automated, unbiased quality testing.

Direct Observation of Translocation in Individual DNA Polymerase Complexes

Background: DNA polymerases translocate along DNA by one nucleotide in each catalytic cycle. Results: The DNA polymerase translocation step is observed with single nucleotide and submillisecond precision. Conclusion: DNA polymerase complexes fluctuate between pre- and post-translocation states and are rectified to the post-translocation state by dNTP. Significance: These results provide insight into the translocation mechanism and its integration into the DNA polymerase catalytic pathway. Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.