Surface Plasmon Biosensor Research Articles

Biosensor technology for real-time detection of the cystic fibrosis W1282X mutation in CFTR.

In the present paper, biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect the Trp1282Ter mutation (W1282X) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene. We first immobilized on a SA5 sensor chip a single-stranded biotinylated oligonucleotide containing the sequence involved in this mutation, and the efficiency of hybridization of oligonucleotide probes differing in length was determined. Second, we immobilized on different SA5 sensor chips biotinylated polymerase-chain reaction (PCR) products from a normal subject as well as from heterozygous and homozygous W1282X samples. The results obtained show that both allele-specific 10- and 12-mer oligonucleotides are suitable probes to detect W1282X mutations of the cystic fibrosis gene under standard BIA experimental conditions. During the association phase performed at 25 degrees C, discrimination between mismatched and full matched hybrids was readily and reproducibly observed by using the 10-mer W1282X probes. By contrast, when the 12-mer DNA probes were employed, discrimination between mismatched and full matched hybrids was observed during the dissociation phase. Taken together, the results presented suggest that BIA is an easy, speedy, and automatable approach to detect point mutations leading to cystic fibrosis. By this procedure, it is possible to perform real-time monitoring of hybridization between target single stranded PCR products obtained by using as substrates DNA isolated from normal or heterozygous subjects, and homozygous W1282X CF samples and oligonucleotide probes, therefore enabling a one-step, non-radioactive protocol to perform diagnosis.

Surface plasmon resonance for real-time monitoring of molecular interactions between a triple helix forming oligonucleotide and the Sp1 binding sites of human Ha-ras promoter: effects of the DNA-binding drug chromomycin.

DNA-binding molecules have been recently proposed as potential inhibitors of molecular interactions between transcription factors and target DNA sequences. Among DNA-binding drugs, chromomycin binds to GC-rich sequences of the Sp1 binding sites of the Ha-ras oncogene. These sites are also molecular targets of a triple-helix forming oligonucleotide [Sp1(Ha-ras)TFO] which is able to inhibit Ha-ras oncogene transcription. We studied molecular interactions between triple-helix forming oligonucleotides and target Sp1 binding sites of the human Ha-ras promoter in the presence of the DNA-binding drug chromomycin. This study was performed by (a) surface plasmon resonance and biosensor technology, (b) gel retardation assay and (c) magnetic capturing of molecular complexes between TFO, chromomycin and target DNA. The main conclusion of our study is that low concentrations of chromomycin allow binding of the triplex-forming oligonucleotide to Sp1 target DNA sequences of the Ha-ras oncogene promoter. Higher concentrations of this DNA-binding drug fully suppress molecular interactions between the Sp1(Ha-ras)TFO and target DNA. Additionally, low concentrations of chromomycin potentiate the effects of the Sp1(Ha-ras)TFO in inhibiting the molecular interactions between purified Sp1 transcription factor and target DNA sequences.

Dissociation rate of antibody-gp120 binding interactions is predictive of V3-mediated neutralization of HIV-1.

mAbs specific for the V3 loop of HIV-1 are capable of neutralizing laboratory strains of HIV-1 in vitro. In this report we use surface plasmon resonance and biosensor technology to demonstrate that the ability of V3-specific mAbs to neutralize HIV-1(MN) correlated with the dissociation rate constant of the homologous mAb-gp120 binding interaction. mAbs capable of binding diverse strains of gp120 with similar association rate constants exhibited marked differences in the dissociation rate. The dissociation rate, and not the association rate, was found to be predictive of the neutralization capacity of the mAb. Furthermore, synthetic peptides corresponding to the V3 loop of HIV-1(IIIB, MN) yielded quantitatively similar binding kinetic profiles abrogating the need for purified recombinant gp120 protein and potentially facilitating the screening of more diverse isolates. Biosensor immobilized V3 peptides were found to mimic their conformational structure in solution. This offers advantages to peptides studied by ELISA where some degree of denaturation and masking of epitopes can occur upon adsorption of peptides to plastic surfaces. The impact of amino acid substitutions within epitopes on subsequent mAb binding was dissected by observing alterations in dissociation rates. The technique provides rapid kinetic analyses of V3 Ab binding interactions with diverse V3 sequences, facilitating the efficient screening and selection of those most likely to possess the broadest and most potent HIV-1 neutralizing potentials.

95 Real-time characterization of the interaction of HIV-1-specific inhibitors with their binding site at the HIV-1 reverse transcriptase (RT) using surface plasmon resonance and biosensor technology: R. Pauwels 1, B. Persson 2, L.G. Fägerstam 2, L. Öfås 2, K. De Vreese 1 P. Van Daele 3, Z. Debyser 1, R. Bhikhabhai 4, B. Strandberg 4, J. Desmyter 1, P.A.J. Janssen

95 Real-time characterization of the interaction of HIV-1-specific inhibitors with their binding site at the HIV-1 reverse transcriptase (RT) using surface plasmon resonance and biosensor technology: R. Pauwels 1, B. Persson 2, L.G. Fägerstam 2, L. Öfås 2, K. De Vreese 1 P. Van Daele 3, Z. Debyser 1, R. Bhikhabhai 4, B. Strandberg 4, J. Desmyter 1, P.A.J. Janssen