| The Adsorption of Bacteriophage ϕX174 to its Host Biophysical Journal, Volume 2, Issue 6, 1 November 1962, Pages 433-449 Robert Fujimura and Paul Kaesberg Abstract The adsorption of purified ϕX174 to C and to C cell walls was investigated. Adsorption was measured by assaying for unadsorbed plaque formers. The amount of irreversible and reversible adsorption depends upon pH and divalent ion concentration. Maximum irreversible adsorption occurs in 0.1 M CaCl at 36°C. There is no detectable reversible adsorption at conditions of pH and CaCl concentration optimum for irreversible adsorption. Under these optimum conditions, diffusion is not the rate-limiting factor, and the encounter efficiency appears to be low. The rate constant is 1.0×10 ml/sec. Phages adsorbed irreversibly to live cells cause infection and to the isolated cell walls apparently cause release of DNA. There is a specific ϕX174 receptor site on the mucocomplex portion of the cell wall. Abstract | PDF (1005 kb) |
| Optical Study of DNA Surface Hybridization Reveals DNA Surface Density as a Key Parameter for Microarray Hybridization Kinetics Biophysical Journal, Volume 92, Issue 3, 1 February 2007, Pages 999-1004 Wolfgang Michel, Timo Mai, Thomas Naiser and Albrecht Ott Abstract 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. Abstract | Full Text | PDF (134 kb) |
| Immobilization of Nucleic Acids at Solid Surfaces: Effect of Oligonucleotide Length on Layer Assembly Biophysical Journal, Volume 79, Issue 2, 1 August 2000, Pages 975-981 A.B. Steel, R.L. Levicky, T.M. Herne and M.J. Tarlov Abstract This report investigates the effect of DNA length and the presence of an anchoring group on the assembly of presynthesized oligonucleotides at a gold surface. The work seeks to advance fundamental insight into issues that impact the structure and behavior of surface-immobilized DNA layers, as in, for instance, DNA microarray and biosensor devices. The present study contrasts immobilization of single-stranded DNA (ssDNA) containing a terminal, 5′ hexanethiol anchoring group with that of unfunctionalized oligonucleotides for lengths from 8 to 48 bases. Qualitatively, the results indicate that the thiol anchoring group strongly enhances oligonucleotide immobilization, but that the enhancement is reduced for longer strand lengths. Interestingly, examination of the probe coverage as a function of strand length suggests that adsorbed thiol-ssDNA oligonucleotides shorter than 24 bases tend to organize in end-tethered, highly extended configurations for which the long-term surface coverage is largely independent of oligonucleotide length. For strands longer than 24 bases, the surface coverage begins to decrease notably with probe length. The decrease is consistent with a less ordered arrangement of the DNA chains, presumably reflecting increasingly polymeric behavior. Abstract | Full Text | PDF (153 kb) |
Copyright © 1995 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 69, Issue 6, 2243-2255, 1 December 1995
doi:10.1016/S0006-3495(95)80095-0
Research Article
V. Chan, D.J. Graves and S.E. McKenzie
Department of Chemical Engineering, University of Pennsylvania, Philadelphia, USA.
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.