Adsorption Kinetics and Ligand-Binding Properties of Thiol-Modified Double-Stranded DNA on a Gold Surface

Abstract

The development of DNA-based biosensors requires efficient immobilization of DNA probes on the sensor surfaces with optimum coverage and orientation. In this study, we have prepared thin films of a 718 base pair 5‘-thiol-labeled double-stranded DNA (dsDNA) on a gold surface by chemisorption and determined the quantity, surface coverage, and adsorption kinetics of dsDNA using cyclic voltammetry (CV), quartz crystal resonator (QCR), and radioactive labeling techniques. The adsorption and desorption rate constants of the 5‘-thiol-labeled dsDNA on a gold surface are estimated to be (1.9 ± 0.2) × 103 M-1 s-1 and (1.3 ± 1.1) × 10-3 s-1, respectively. The equilibrium constant is (1.5 ± 1.3) × 106 M-1 and the fractional coverage is >75% for the DNA concentration range of 2−10 μM. The ligand-binding properties of the immobilized dsDNA were investigated using doxorubicin, a DNA-intercalating anthracyclic antibiotic, as the probing molecule. The binding of doxorubicin to the immobilized dsDNA was examined by CV to verify the ligand-binding capacity and specificity of the immobilized dsDNA. The kinetics of the interaction between doxorubicin conjugated with a 200-kDa dextran polymer and the immobilized dsDNA was determined by QCR, which gives the association and dissociation rate constants of (1.8 ± 0.5) × 103 M-1 s-1 and (3.5 ± 1.5) × 10-3 s-1, respectively, and an equilibrium binding constant of (5.1 ± 2.6) × 105 M-1. The binding parameters for the conjugate−DNA interaction were also determined by using a surface plasmon resonance (SPR)-based biosensor, in which dsDNA was immobilized on the hydrogel matrix of the sensor chip through a biotin−streptavidin linkage. While the kinetic constants from SPR measurements, k a = (5.7 ± 0.2) × 103 M-1 s-1 and k d = (1.2 ± 0.2) × 10-2 s-1, are different from those determined by QCR, the thermodynamic constant K A obtained by SPR, (4.8 ± 0.8) × 105 M-1, is similar to that by QCR.

Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors

“…The peak currents of the redox couple decreased and the peak-to-peak separation ( E p ) increased when probe-modified electrodes were used and compared with bare electrodes. The shielding of the ferro-ferri-cyanide ions from the DNA-modified surface was attributed to a combination of physical coverage by the ssDNA and the electrostatic repulsion between the negatively charged redox couple ions and the DNA phosphate backbone (Yang et al, 1998). Takenaka et al (2000) reported an indirect quantification method based on the HPLC analysis of the excess of DNA remained unadsorbed.…”

DNA Electrochemical Behaviors, Recognition and Sensing by Combining with PCR Technique

“…Up to now, carbon materials including carbon paste, graphite, glassy carbon, carbon fiber, carbon nanotube and screen-printed electrodes been widely used for the nucleic acid research and electrochemical detection due to their several advantages such as easy handle, renewable, cheap, convenient and disposable in coming commercial uses . Various metals or metal oxides such as gold [77][78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94][95][96], platinum [97,98], silver [99][100][101] and indium tin oxide [102,103] etc have also been used in this filed.…”

“…Recently Paleček et al extended the choice of hybridization surfaces and detection electrodes [28,29,136]. Although label-free strategies for DNA recognition [137] and mediated electron-transfer for DNA detection [138,139] have become a promising aspect, the study of DNA hybridization biosensors using simple intercalators, threading intercalators or bisintercalators as redox indicators is still an important area [11,12,17,59,62,78,79,80,85,87,92,107,110,140]. Besides the intercalating, the interaction between DNA and electroactive indicator may involve the electrostatic [30,78,79,91,107] or groove [110] mechanism.…”

Mechanism of active targeting in solid tumors with transferrin-containing gold nanoparticles

“…The decoration of AuNPs with thiolated poly(ethylene glycol) (PEG-SH) via the formation of stable, covalent gold-thiol linkages (bond energy = 30-40 kJ/mol) (19) can reduce nonspecific interactions (20), allowing the targeting ligands immobilized on the particle surface to engage cell surface receptors with high specificity. PEGylation also prolongs the circulation time of nanoparticles (21), giving sufficient time for particles to localize in different organs.…”