Optical study of DNA surface hybridization reveals DNA surface density as a key parameter for microarray hybridization kinetics

We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22 mer strand (bound-DNA) is immobilized via phenylene-diisothiocyanate linker molecule on the substrate, the dye-labeled (Cy3) complementary strand (free-DNA) is in solution in a reaction chamber. We use total internal reflection fluorescence for surface detection of hybridization. As a new feature we perform a simultaneous real-time measurement of the change of free-DNA concentration in bulk parallel to the total internal reflection fluorescence measurement. We observe that the free-DNA concentration decreases considerably during hybridization. We show how the standard Langmuir kinetics needs to be extended to take into account the change in bulk concentration and explain our experimental results. Connecting both measurements we can estimate the surface density of accessible, immobilized bound-DNA. We discuss the implications with respect to DNA microarray detection.     

DNA immobilization/hybridization on plasma-polymerized pyrrole

The present work describes the fabrication and characterization of the conducting polymer coatings prepared by the continuous wave plasma polymerization and the applications as adhesion layers for studying DNA immobilization/hybridization. The stability of plasma polymerized pyrrole (ppPY) in the aqueous solution was characterized by ellipsometry. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were used to investigate polymer matrix properties and oligonucleotide/DNA binding interaction. The successful DNA immobilization on ppPY surfaces was found to depend on the macromolecular architecture of plasma polymerized films. The plasma polymers with similar thickness deposited at different input powers showed various comparable immobilization properties. The plasma-polymerized films prepared at the low input power showed a lower sensitivity toward DNA binding than those films deposited at the high input power. This result will be important to study plasma polymerized films as potential DNA biosensors in the future.     

Mass transfer effects on DNA hybridization in a flow-through microarray

The goal of this study is to assess the influence of mass transfer phenomena on DNA hybridization kinetics in a flow-through, porous microarray for fast molecular testing. We present a scaled mathematical model of coupled convection, diffusion and reaction in porous media, which was used to simulate hybridization kinetics and to analyze the influence of convective transport on the reaction rate. In addition to computer simulations, we also present experimental data of hybridization collected on our microarray system for different flow rates. The results reported in this paper provide for a better understanding of the interaction between reaction and mass transfer processes during flow-through hybridization and suggest criteria for system design and optimization.     

Frequency dependent and surface characterization of DNA immobilization and hybridization

The hybridization of oligomeric DNA was investigated using the frequency dependent techniques of quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS). Synthetic 5'-amine-terminated single stranded oligonucleotides (ssDNA) were immobilized on the surface of the oxidized platinum driving electrodes of AT-cut quartz QCM crystals using 3-glycidoxypropyl-trimethoxysilane. Similar ssDNA coupling was accomplished on the exposed glass surface between the metallic digits of microlithographically fabricated interdigitated microsensor electrodes (IMEs). Confirmation of this covalent coupling surface chemistry was achieved using Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR). Substantial changes in resonant frequency values (0.012% decrease) and electrochemical impedance values (both real and imaginary components) (35.4 and 42.1% increase in impedance magnitude at 1.0 Hz in buffer and deionized water, respectively) were observed resulting from hybridization of the attached ssDNA upon exposure to its complement under appropriate hybridization conditions. Non-complementary (random) oligomer sequence demonstrated a modest change in resonant frequency and a non-detectable change in impedance. Microarray glass slide surfaces modified with 3-glycidoxypropyltrimethoxysilane (GPS), shown to be advantageous in the design and use of microarrays of amine-terminated ssDNA, is confirmed to arise from direct covalent coupling of the DNA to the surface with little non-specific adsorption. The possibility to detect the binding state of DNA in the vicinity of an electrode, without a direct connection between the measurement electrode and the DNA is hereby reported. The potential for development of label-free, low-density DNA microarrays is demonstrated and is being pursued.     

Effect of formaldehyde on the efficiency of hybridization of DNA immobilized on nitrocellulose filters.

We report in this study that under certain conditions formaldehyde interacts with DNA and makes it more efficient for hybridization on nitrocellulose filters. Hybridization signals of formaldehyde-treated DNA are stronger (up to 10 fold) as compared with that of the heat- or alkali-denatured DNA. Various parameters of the DNA-formaldehyde reaction are optimized as follows: (a) 6 x SSC, 10% formaldehyde, 60 degrees C, 20-30 min, reaction volume 10-200 microliters or (b) 6 x SSC, 5% formaldehyde, 98 degrees C, 15 min, reaction volume 10-200 microliters. Treatment of agarose gels after electrophoresis with formaldehyde improved both the transfer of DNA and the efficiency of hybridization. The following conditions are recommended for gel treatment: denaturation in 0.3 N NaOH, 1 M NaCl followed by neutralization with 0.5 M phosphate buffer, pH 7.0, containing 10% formaldehyde at 60 degrees C for 20 min.     

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.     

Chromatin fibers are left-handed double helices with diameter and mass per unit length that depend on linker length

Four classes of models have been proposed for the internal structure of eukaryotic chromosome fibers--the solenoid, twisted-ribbon, crossed-linker, and superbead models. We have collected electron image and x-ray scattering data from nuclei, and isolated chromatin fibers of seven different tissues to distinguish between these models. The fiber diameters are related to the linker lengths by the equation: D(N) = 19.3 + 0.23 N, where D(N) is the external diameter (nm) and N is the linker length (base pairs). The number of nucleosomes per unit length of the fibers is also related to linker length. Detailed studies were done on the highly regular chromatin from erythrocytes of Necturus (mud puppy) and sperm of Thyone (sea cucumber). Necturus chromatin fibers (N = 48 bp) have diameters of 31 nm and have 7.5 +/- 1 nucleosomes per 10 nm along the axis. Thyone chromatin fibers (N = 87 bp) have diameters of 39 nm and have 12 +/- 2 nucleosomes per 10 nm along the axis. Fourier transforms of electron micrographs of Necturus fibers showed left-handed helical symmetry with a pitch of 25.8 +/- 0.8 nm and pitch angle of 32 +/- 3 degrees, consistent with a double helix. Comparable conclusions were drawn from the Thyone data. The data do not support the solenoid, twisted-ribbon, or supranucleosomal particle models. The data do support two crossed-linker models having left-handed double-helical symmetry and conserved nucleosome interactions.     

Enhancement of DNA immobilization and hybridization on gold electrode modified by nanogold aggregates

Gold electrodes modified by nanogold aggregates (nanogold electrode) were obtained by the electrodeposition of gold nanoparticles onto planar gold electrode. The Electrochemical response of single-stranded DNA (ssDNA) probe immobilization and hybridization with target DNA was measured by cyclic voltammograms (CV) using methylene blue (MB) as an electroactive indicator. An improving method using long sequence target DNA, which greatly enhanced the response signal during hybridization, was studied. Nanogold electrodes could largely increase the immobilization amount of ssDNA probe. The hybridization amount of target DNA could be increased several times for the manifold nanogold electrodes. The detection limit of nanogold electrode for the complementary 16-mer oligonucleotide (target DNA1) and long sequence 55-mer oligonucleotide (target DNA2) could reach the concentration of 10(-9) mol/L and 10(-11) mol/L, respectively, which are far more sensitive than that of the planar electrode.     

Improved immobilization of DNA to microwell plates for DNA-DNA hybridization.

An improved and simplified protocol for DNA immobilization was developed to enhance DNA-DNA hybridization on microwell plates. Target DNA was immobilized by simple dry-adsorption. Efficiencies of DNA immobilization and retention were enhanced 1.4-6.5 times and 4.2-19.6 times, respectively, compared with a conventional method. The overall hybridization efficiency was increased 3.1-5.2 times. This simple new protocol can reduce the consumption of scarce DNA samples.     

Optimization of the BLASTN substitution matrix for prediction of non-specific DNA microarray hybridization

DNA microarray measurements are susceptible to error caused by non-specific hybridization between a probe and a target (cross-hybridization), or between two targets (bulk-hybridization). Search algorithms such as BLASTN can quickly identify potentially hybridizing sequences. We set out to improve BLASTN accuracy by modifying the substitution matrix and gap penalties. We generated gene expression microarray data for samples in which 1 or 10% of the target mass was an exogenous spike of known sequence. We found that the 10% spike induced 2-fold intensity changes in 3% of the probes, two-third of which were decreases in intensity likely caused by bulk-hybridization. These changes were correlated with similarity between the spike and probe sequences. Interestingly, even very weak similarities tended to induce a change in probe intensity with the 10% spike. Using this data, we optimized the BLASTN substitution matrix to more accurately identify probes susceptible to non-specific hybridization with the spike. Relative to the default substitution matrix, the optimized matrix features a decreased score for A–T base pairs relative to G–C base pairs, resulting in a 5–15% increase in area under the ROC curve for identifying affected probes. This optimized matrix may be useful in the design of microarray probes, and in other BLASTN-based searches for hybridization partners.     

Modeling of DNA hybridization kinetics for spatially resolved biochips

The marriage of microfluidics with detection technologies that rely on highly selective nucleic acid hybridization will provide improvements in bioanalytical methods for purposes such as detection of pathogens or mutations and drug screening. The capability to deliver samples in a controlled manner across a two-dimensional hybridization detection platform represents a substantial technical challenge in the development of quantitative and reusable biochips. General theoretical and numerical models of heterogeneous hybridization kinetics are required in order to design and optimize such biochips and to develop a quantitative method for online interpretation of experimental results. In this work we propose a general kinetic model of heterogeneous hybridization and develop a technique for estimating the kinetic coefficients for the case of well-spaced, noninteracting surface-bound probes. The experimentally verified model is then incorporated into the BLOCS (biolab-on-a-chip simulation) 3D microfluidics finite element code and used to model the dynamic hybridization on a biochip surface in the presence of a temperature gradient. These simulations demonstrate how such a device can be used to discriminate between fully complementary and single-base-pair mismatched hybridization using fluorescence detection by interpretation of the unique spatially resolved intensity pattern. It is also shown how the dynamic transport of the targets is likely to affect the rate and location of hybridization as well as that, although nonspecific hybridization is present, the change in the concentration of hybridized targets over the sensor platform is sufficiently high to determine if a fully complementary match is present. Practical design information such as the optimum transport speed, target concentration, and channel height is presented. The results presented here will aid in the interpretation of results obtained with such a temperature-gradient biochip.     

Method for estimating the kinetics of substrate degradation in horizontal-flow anaerobic immobilized sludge reactors

Apparent and intrinsic kinetic parameters of substrate decomposition were estimated from the profiles of chemical oxygen demand (COD) and volatile fatty acids (VFA) concentrations along the length of the horizontal-flow anaerobic immobilized sludge (HAIS) reactor. The first order substrate utilization kinetic model describes well the experimental data.     

Influence of secondary structure on kinetics and reaction mechanism of DNA hybridization

Hybridization of nucleic acids with secondary structure is involved in many biological processes and technological applications. To gain more insight into its mechanism, we have investigated the kinetics of DNA hybridization/denaturation via fluorescence resonance energy transfer (FRET) on perfectly matched and single-base-mismatched DNA strands. DNA hybridization shows non-Arrhenius behavior. At high temperature, the apparent activation energies of DNA hybridization are negative and independent of secondary structure. In contrast, when temperature decreases, the apparent activation energies of DNA hybridization change to positive and become structure dependent. The large unfavorable enthalpy of secondary structure melting is compensated for by concomitant duplex formation. Based on our results, we propose a reaction mechanism about how the melting of secondary structure influences the hybridization process. A significant point in the mechanism is that the rate-limiting step switches along with temperature variation in the hybridization process of structured DNA, because the free energy profile of hybridization in structured DNA varies with the variation in temperature.     

Quantitative analysis of DNA hybridization in a flowthrough microarray for molecular testing

Quantitative information about the nucleic acids hybridization reaction on microarrays is fundamental to designing optimized assays for molecular diagnostics. This study presents the kinetic, equilibrium, and thermodynamic analyses of DNA hybridization in a microarray system designed for fast molecular testing of pathogenic bacteria. Our microarray setup uses a porous, nylon membrane for probe immobilization and flowthrough incubation. The Langmuir model was used to determine the reaction rate constants of hybridization with antisense targets specific to Staphylococcus epidermidis and Staphylococcus aureus strains. The kinetic analysis revealed a sequence-dependent reaction rate, with association rate constants on the order of 10⁵ M⁻¹ s⁻¹ and dissociation rate constants of 10⁻⁴ s⁻¹. We found that by increasing the probe surface density from 10¹¹ to 10¹² molecules/cm², the hybridization rate and efficiency are suppressed while the melting temperature of the DNA duplex increases. The maximum fraction of hybridized capture probes at equilibrium did not exceed 50% for hybridization with antisense sequences and was below 6% for hybridization with long targets obtained from PCR. The van’t Hoff analysis of the temperature denaturation data showed that the DNA hybridization in our porous, flowthrough microarray is thermodynamically less favorable than the hybridization of the same sequences in solution.     

Metabolism and growth in Arabidopsis depend on the daytime temperature but are temperature-compensated against cool nights

Diurnal cycles provide a tractable system to study the response of metabolism and growth to fluctuating temperatures. We reasoned that the response to daytime and night temperature may vary; while daytime temperature affects photosynthesis, night temperature affects use of carbon that was accumulated in the light. Three Arabidopsis thaliana accessions were grown in thermocycles under carbon-limiting conditions with different daytime or night temperatures (12 to 24 °C) and analyzed for biomass, photosynthesis, respiration, enzyme activities, protein levels, and metabolite levels. The data were used to model carbon allocation and growth rates in the light and dark. Low daytime temperature led to an inhibition of photosynthesis and an even larger inhibition of growth. The inhibition of photosynthesis was partly ameliorated by a general increase in protein content. Low night temperature had no effect on protein content, starch turnover, or growth. In a warm night, there is excess capacity for carbon use. We propose that use of this capacity is restricted by feedback inhibition, which is relaxed at lower night temperature, thus buffering growth against fluctuations in night temperature. As examples, the rate of starch degradation is completely temperature compensated against even sudden changes in temperature, and polysome loading increases when the night temperature is decreased.     

Comparison of substrate utilization and growth kinetics between immobilized and suspended Pseudomonas cells

A methodology is described for measurement if immobilized and suspended cell growth and substrate utilization kinetics parameters. Substrate utilization and growth kinetics were compared between immobilized and suspended cells for toluene degrading Pseudomonas strains K3-2 and 2,4-dichlorophenoxyacetic acid (2,4-D) degrading strain DBO131(pR0101), respectively. Kinetic parameters were estimated using nonlinear parameter estimation methods and compared between the immobilized and suspended Pseudomonas cells to determine the effect of immobilization on cellular growth and substrate utilization. Factors influencing the experimental design included calculated oxygen flux rates, primary carbon substrate flux rates, and shear stresses on the immobilize cell. Statistical interpretation of the cellular reaction rate parameters indicates that only the growth kinetics of the toluene system were significantly altered upon immobilization. Substrate utilization kinetics remained unchanged upon immobilization. The substrate growth associated half-saturation constant (K(g)) for the toluene system increased by 30-fold and the maximum specific growth rate (mu(max)) decreased by 2-fold upon immobilization. Implication of these results for experimental determination of cellular kinetic parameters and for immobilization cell bioreactors design are discussed. (c) 1993 John Wiley & Sons, Inc.     

Stabilization of the immobilized linkers and DNA probes for DNA microarray fabrication by end-capping of the remaining unreacted silanol on the glass

High stability of the oligonucleotides immobilized on the glass is essential for the reliable DNA microarray analysis. In the present study, effect of end-capping of the unreacted silanol, remaining after the surface amine-functionalization, was explored: (1) Cy3-NHS (N-hydorxysuccinincimide) dye was spotted on the surface and change in the fluorescent intensity was measured. (2) DNA probes were immobilized by the reactivity of oxanine linked at the 5’-end, the complementary oligonucleotides with Cy5-fluorescence at the 5’-end was hybridized, and the time-dependence of the fluorescence intensity was observed. Both the systems showed improved stability of the immobilized molecules, indicative of the stabilization by end-capping.