DNA binding and hybridization on gold and derivatized surfaces
ABSTRACT The binding of various single- and double-stranded DNAs onto gold and derivatized gold surfaces, and their hybridization with complementary DNA species, have been investigated using a quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). The DNA species employed were a 21-mer oligonucleotide (Mbo21), several double-stranded plasmid DNAs (7.2 kilobases) modified by incorporation of α-phosphothio-nucleotides into the ends of the linearized plasmid DNA (pPS-Sx, where x represents the number of α-phosphothio-nucleotides), and a 30-mer oligonucleotide having a mercaptohexyl group at the 5′-phosphate end (BS1-SH). Both QCM and SPR data reveal that unmodified DNA does not spontaneously adsorb onto underivatized gold surfaces from aqueous solutions. Modification of the gold surface through the attachment of an ionizable thiol compound, 2-dimethylaminoethanethiol hydrochloride (DMAET), allows DNA to adsorb through electrostatic interactions. SPR measurements confirm the presence of Mbo21 DNA on the DMAET-modified gold surface. Immobilized Mbo21, however, does not undergo hybridization. QCM and SPR data suggest that pPS-S4, pPS-S50 and BS1-SH DNA all assume a flat orientation on gold. No hybridization of single-stranded DNA to gold-immobilized pPS-S4 and pPS-S50 could be detected. In contrast 30-mer DNA binding from solution to the complement BS1-SH immobilized on gold reveals hybridization of the DNA strands.
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ABSTRACT: A comparison between the quartz-crystal microbalance (QCM) and surface plasmon resonance (SPR) using an especially designed flow-through cell for simultaneous measurements and flow-injection analysis (FIA) is presented. We compare the effects due to unspecific binding of bovine serum albumin (BSA) to gold surfaces, specific binding between BSA and monoclonal antibodies, regeneration experiments, serum experiments with HIV antigens and the influence of the bulk properties of liquid samples. The basic theory and its consequences on measurement signals and experimental methods are discussed. The fundamental problem of the comparability of different measurement principles is considered. We suggest guide parameters, which allow an objective judgement of the performance. We do not find any advantage of one particular system concerning sensitivity and cross sensitivity.Sensors and Actuators B: Chemical. 01/1995;
- Biopolymers 02/1970; 9(9):1029-38. · 2.88 Impact Factor
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ABSTRACT: The effect of flow rate on the binding of polyclonal sheep immunoglobulin G (IgG) and anti-sheep IgG to gold, and to immobilized anti-IgG and IgG receptor layers, respectively, has been studied using the optical technique of surface plasmon resonance at a gold-solution interface. The kinetics of protein binding were investigated by monitoring the reflectivity of light at a fixed angle close to the plasmon resonance. Fixed-angle reflectivity data are presented in terms of protein surface coverage. IgG and anti-IgG effective thicknesses were determined by fitting the full angle-dependent reflectivity data to Fresnel theory. The effective thicknesses of IgG and anti-IgG bound to the gold surface and to each other (as pre-adsorbed layers) were found to be independent of the flow rate of protein solution. Gold-adsorbed IgG and anti-IgG layers were found to be 63 ± 15 Å and 68 ± 10 Å thick, respectively, and 91 ± 11 Å (anti-IgG) and 20 ± 7 Å (IgG) thick when subsequently bound to their biospecific partner. The differences in the thickness values between the gold-adsorbed and protein-adsorbed layers are explained in terms of the orientation of the immobilized proteins and their deformation upon binding to the gold surface. Non-specific binding between immobilized sheep IgG layers and anti-mouse IgG was shown to be negligible.Colloids and Surfaces A-physicochemical and Engineering Aspects - COLLOID SURFACE A. 01/1995; 103(3):147-157.