Rastislav Levicky

DNA surface hybridization regimes

Surface hybridization reactions, in which sequence-specific recognition occurs between immobilized and solution nucleic acids, are routinely carried out to quantify and interpret genomic information. Although hybridization is fairly well understood in bulk solution, the greater complexity of an interfacial environment presents new challenges to a fundamental understanding, and hence application, of these assays. At a surface, molecular interactions are amplified by the two-dimensional nature of the immobilized layer, which focuses the nucleic acid charge and concentration to levels not encountered in solution, and which impacts the hybridization behavior in unique ways. This study finds that, at low ionic strengths, an electrostatic balance between the concentration of immobilized oligonucleotide charge and solution ionic strength governs the onset of hybridization. As ionic strength increases, the importance of electrostatics diminishes and the hybridization behavior becomes more complex. Suppression of hybridization affinity constants relative to solution values, and their weakened dependence on the concentration of DNA counterions, indicate that the immobilized strands form complexes that compete with hybridization to analyte strands. Moreover, an unusual regime is observed in which the surface coverage of immobilized oligonucleotides does not significantly influence the hybridization behavior, despite physical closeness and hence compulsory interactions between sites. These results are interpreted and summarized in a diagram of hybridization regimes that maps specific behaviors to experimental ranges of ionic strength and probe coverage.

Real-time, multiplexed electrochemical DNA detection using an active complementary metal-oxide-semiconductor biosensor array with integrated sensor electronics

Optical biosensing based on fluorescence detection has arguably become the standard technique for quantifying extents of hybridization between surface-immobilized probes and fluorophore-labeled analyte targets in DNA microarrays. However, electrochemical detection techniques are emerging which could eliminate the need for physically bulky optical instrumentation, enabling the design of portable devices for point-of-care applications. Unlike fluorescence detection, which can function well using a passive substrate (one without integrated electronics), multiplexed electrochemical detection requires an electronically active substrate to analyze each array site and benefits from the addition of integrated electronic instrumentation to further reduce platform size and eliminate the electromagnetic interference that can result from bringing non-amplified signals off chip. We report on an active electrochemical biosensor array, constructed with a standard complementary metal-oxide-semiconductor (CMOS) technology, to perform quantitative DNA hybridization detection on chip using targets conjugated with ferrocene redox labels. A 4 x 4 array of gold working electrodes and integrated potentiostat electronics, consisting of control amplifiers and current-input analog-to-digital converters, on a custom-designed 5 mm x 3 mm CMOS chip drive redox reactions using cyclic voltammetry, sense DNA binding, and transmit digital data off chip for analysis. We demonstrate multiplexed and specific detection of DNA targets as well as real-time monitoring of hybridization, a task that is difficult, if not impossible, with traditional fluorescence-based microarrays.

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.

Active CMOS Array Sensor for Time-Resolved Fluorescence Detection

Surface-based sensing assays based on fluorescence-based detection have become commonplace for both environmental and biomedical diagnostics. Traditional array scanners are expensive, large, and complex instruments. This paper describes the design of an active CMOS biosensor substrate for fluorescence-based assays that enables time-gated, time-resolved fluorescence spectroscopy without the need for an external reader. The array is sensitive to photon densities as low as 1.15times108/cm2, has a dynamic range of over 74 dB, and has subnanosecond timing resolution. Sensitivity is achieved through subsampling and averaging

Distance control for a near-field scanning microwave microscope in liquid using a quartz tuning fork

We demonstrate a scanning near-field microwave microscope (NSMM) in the liquid environment using a tuning fork shear-force feedback method to control the distance between tip and sample. Only the probe tip for the NSMM is immersed in water. The dry part of the probe is attached to one prong of a quartz tuning fork and directly coupled to a high-quality dielectric resonator at an operating frequency f=4.5–5.5GHz. This distance control method is independent of the local microwave characteristics. The amplitude of the tuning fork was used as a set point of the distance control parameter in the liquid. To demonstrate the distance regulation system, we present the NSMM images of a copper film in air and liquid without and with readjustment of the distance set point, as well as an image of a DNA film in buffer solution. Imaging under buffer environments is of particular interest for future studies of biomolecular association reactions on solid supports.

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This paper describes the design of an active, integrated CMOS sensor array for fluorescence applications which enables time-gated, time-resolved fluorescence spectroscopy. The 64-by-64 array is sensitive to photon densities as low as 8.8 times 106 photons/cm2 with 64-point averaging and, through a differential pixel design, has a measured impulse response of better than 800 ps. Applications include both active microarrays and high-frame-rate imagers for fluorescence lifetime imaging microscopy.

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Five electroactive ferrocene tags for labelling of biomolecules are contrasted with regard to conjugation reactivity with amine and thiol moieties, stability to loss of electrochemical activity, and impact of molecular structure on the redox potential of the free and DNA-conjugated forms.

m CMOS Array Sensor for Integrated Time-Resolved Fluorescence Detection

DNA microarrays have proven extraordinarily powerful for differential expression studies across thousands of genes in a single experiment. Microarrays also have the potential for clinical applications, including the detection of infectious and immunological diseases and cancer, if they can be rendered both reliable and cost-effective. Here we report the first practical application of an active microarray based on integrated circuit technology, completely obviating the need for external measurement instrumentation while employing protocols compatible with traditional fluorescence-based surface bioassays. In a gene expression biodosimetry study, we determine the differential activity of genes from leucocytes in irradiated human blood. Quantum dots are used as fluorescence labels to realize filterless, time-gated fluorescence detection on an active complementary metal-oxide-semiconductor (CMOS) microarray with 100-pM sensitivity. Improvements in surface chemistry should allow sensitivities that approach the microarray hardware limit of less than 10 pM. Techniques for covalent attachment of DNA capture strands to the CMOS active microarrays allow integrated sensors to be placed in immediate proximity to hybridized analyte strands, maximizing photon collection efficiencies.

A comparison of five bioconjugatable ferrocenes for labeling of biomolecules

UNLABELLED Electrokinetic methods that conveniently concentrate charged analytes by orders of magnitude are highly attractive for nucleic acid assays where they can bypass the complexity and costs of enzyme-based amplification. The present study demonstrates an electrokinetic concentration device incorporating charge-neutral morpholino (MO) probes: as DNA analyte is concentrated in a microfluidic channel using ion concentration polarization (ICP) it is simultaneously hybridized to spots of complementary MO probes immobilized on the channel floor. This approach is uniquely favored by the match between the optimum buffer ionic strength of approximately 10mM for both MO-DNA surface hybridization and electrokinetic concentration. The simple and easily scalable poly(dimethylsiloxane) (PDMS) microfluidic device was fabricated using soft lithography and contact printing of a conductive polymer, poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate ( PEDOT PSS) as a cation-selective membrane material. Using the microfluidic concentrator, we could increase the concentration of DNA by three orders of magnitude in less than 5 min at an electric field of 75 Vcm(-1). The 1000-fold increase in concentration of DNA led to an increase in the speed of MO-DNA hybridization by two orders of magnitude and enabled a detection sensitivity of ~1 nM within 15 min of concentration. Using the proposed microfluidic concentrator, we also demonstrated a rapid hybridization with a binary DNA mixture, containing a fully complementary and a non-complementary sequence to mimic molecular backgrounds present in real DNA samples.

Gene expression analysis with an integrated CMOS microarray by time-resolved fluorescence detection

The physical behavior of polyelectrolytes at solid-liquid interfaces presents challenges both in measurement and in interpretation. An informative, yet often overlooked, property that characterizes the equilibrium organization of these systems is their membrane or rest potential. Here a general classification scheme is presented of the relationship between the rest potential and structural response of polyelectrolyte films to salt concentration. A numerical lattice theory, adapted from the polymer community, is used to analyze the rest potential response of end-tethered polyelectrolyte layers in which electrostatics and short-range contact interactions conspire to bring about different structural states. As an experimental quantity the rest potential is a readily accessible, nonperturbing metric of the equilibrium structure of a polyelectrolyte layer. A first set of measurements is reported on monolayers of end-tethered, single-stranded DNA in monovalent (NaCl) and divalent (MgCl(2)) counterion environments. Intriguingly, in NaCl electrolyte at least two different mechanisms appear by which the DNA layers can structurally relax in response to changing salt conditions. In MgCl(2) the layers appear to collapse. The possible molecular mechanisms behind these behaviors are discussed. These studies provide insight into phenomena more generally underlying polyelectrolyte applications in the chemical, environmental, and biotechnological fields.