Biomedical subjects

Danfeng Yao

Publications and source records attributed to Danfeng Yao.

6 recordsLinked to original sources

PubMed2005

Surface density dependence of PCR amplicon hybridization on PNA/DNA probe layers.

Surface plasmon field-enhanced fluorescence spectroscopy was employed to extensively investigate the hybridization behaviors of polymerase chain reaction (PCR) amplicons on a peptide nucleic acid (PNA) or DNA probe layer that was previously attached on a streptavidin-modified gold surface via biotin/streptavidin interaction. Despite the neutral backbone of PNA, the hybridization reactions were strongly influenced by the variation of ionic strength. The association rates exhibited a monotonic decrease with ionic strength increase and the maximum hybridization signal was achieved at an intermediate sodium concentration (approximately 100 mM). These effects were mainly ascribed to the electrostatic cross talk among the hybridized DNA molecules and the secondary structure of PCR amplicons. For the negatively charged DNA probes, the hybridization reaction was subjected additionally to the DNA/DNA electrostatic barrier, particularly in lower ionic strength range (e.g., 10 approximately 150 mM Na(+)). The electrostatic cross talk was shown to be largely reduced if the PNA probe layer was sufficiently diluted by following a strategic templated immobilization method. As a consequence, a pseudo-first-order kinetic model was applicable to describe the hybridization kinetics, and affinity constants were derived for evaluating the influence of single nucleotide polymorphisms (SNPs).

Biophysics↗

PubMed2004

Surface plasmon field-enhanced fluorescence spectroscopy in PCR product analysis by peptide nucleic acid probes.

Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) was recently developed for PCR product analysis, which allowed for real-time monitoring of hybridization processes and for the detection of trace amounts of PCR products, with a detection limit of 100 fmol on the peptide nucleic acid (PNA) probe surface, and 500 fmol on the DNA probe surface. By selectively labeling the strands of PCR-amplified DNA, it was shown that the heat denaturation process in combination with the application of low-salt condition substantially reduced the interference from the antisense strands and thus simplified the surface hybridization. Furthermore, SPFS was demonstrated to be capable of quantitatively discriminating the difference induced by single nucleotide substitution, even within one minute of contact time.

Antisense Elements (Genetics)↗

PubMed2004

Matching base-pair number dependence of the kinetics of DNA-DNA hybridization studied by surface plasmon fluorescence spectroscopy.

Two single-stranded DNA oligonucleotides consisting of complementary base-pairs can form double strands. This phenomenon is well studied in solutions, however, in order to clarify the physical mechanism of the hybridization occurring at a solid/solution interface, we studied the kinetics by surface plasmon fluorescence spectroscopy (SPFS): one single-stranded oligo-DNA (probe-DNA) was immobilized on the substrate, the other one (target-DNA) labelled with a fluorescent probe was added to the flow cell. After hybridization, the chromophores could be excited by the surface plasmon mode and their fluorescence detected with high sensitivity. The dependence of the k(on) and k(off) rate constants on the length of the hybridizing oligonucleotides was investigated by using a MM0 series (no mismatch) and the kinetics was found to be well described by a Langmuir adsorption model. From these measurements we found that also in the case of surface hybridization the affinity of the duplexes decreases as the number of matching base-pairs decreases from 15 to 10. In order to show that SPFS is the powerful technique with high sensitivity, the hybridization process for mixed target-oligos was measured by SPFS and analyzed by an expanded Langmuir model in which two components of target-oligo can bind to probe-DNA at the sensor surface competitively. Two sets of the k(on) and k(off) obtained from the experiment are successfully consistent with the k(on) and k(off) obtained from experiments for single (pure) target-DNA.

Base Pairing↗

PubMed2004

Surface plasmon enhanced diffraction for label-free biosensing.

Surface plasmon enhanced evanescent field at a (noble) metal/dielectric interface can be employed to enhance the diffraction efficiency of surface grating structure composed of biomolecules. Based on a Kretschmann configuration, we realized a diffraction biosensor to monitor the dynamic interaction of biological molecules in a label-free way. It was demonstrated by the binding of an anti-biotin antibody to the biotin-functionalized region of a periodically patterned surface, which generated significant optical contrast to diffract the surface plasmon field. With the aid of the synchronic surface plasmon resonance signal, a quadratic dependence of diffraction signal on the amount of bound antibody was found, which coincides with the theoretical expectation. Time-dependent measurements were conducted to estimate the density of biotin thiols on the functional region.

Antibodies, Monoclonal↗

PubMed2004

Oligonucleotide hybridization studied by a surface plasmon diffraction sensor (SPDS).

A novel label-free biosensor concept based on surface plasmon-enhanced diffraction by micro- patterned interfaces was applied to the study of hybridization reactions of target DNA oligonucleotides (15mers and 75mers) from solution to probe DNA oligonucleotides attached via streptavidin to the sensor surface. The self-referencing and quadratic signal amplification mechanism of the sensor allowed highly sensitive detection of the hybridization process. Association and dissociation processes of DNA targets could be recorded in real time and used for the quantification of their binding affinities, which differ considerably with a single base pair mismatch. An equilibrium titration approach was also applied in order to obtain the binding affinities for 15mer targets, yielding similar affinity values. The hybridization efficiencies were found to be higher for the 15mers than for the 75mers, although the latter contained the same recognition sequences. The hybridization efficiency was shown to depend on the probe density and reached nearly 100% for the 15mer fully complementary targets at a probe density of approximately 1.2 x 10(12) molecules/cm2. Using the assay as an end-point determination method, the lowest detectable coverage of a 15mer oligonucleotide was at least approximately 1.1 x 10(11) molecules/cm2. The diffraction sensing concept offers a completely novel way to integrate a reference channel in large-scale, label-free screening applications, to improve the stability and to enhance the sensitivity of microarray read-out systems.

Base Pair Mismatch↗

PubMed2003

Surface plasmon field-enhanced fluorescence spectroscopy studies of the interaction between an antibody and its surface-coupled antigen.

Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) uses the greatly enhanced electromagnetic field of a surface plasmon mode for the excitation of surface-confined fluorophores. The ability to simultaneously monitor the interfacial refractive index changes and the fluorescence signals in real time offers a huge potential for applications of SPFS in surface immunoreaction detection. In this study, gold surfaces were functionalized by mixed self-assembled monolayers exposing an antigen (biotin) at a density that was varied over a wide range. Specific antibody-antigen interactions were observed for anti-biotin antibody solutions passing over the surfaces with a rather high flow speed driven by a home-built liquid-handling system. First, the use of the fluorophores Cy5 and Alexa Fluor 647 in SFPS-based immunoassays was investigated. It was found that Cy5 exhibits strong self-quenching, which makes it rather unsuitable for quantitative measurements. For the in situ measurement of the binding kinetics, an angular "detuning" effect was confirmed to negatively interfere with the fluorescence signal in cases where large SPR signals were detected. An in-depth comparison between the SPR and the fluorescence signal reveals that the fluorescence yield of the dyes depends strongly on the separation distance from the gold surface. And finally, we stress the ability of SPFS to detect binding to surfaces containing extremely diluted antigen density, where the SPR signal failed to follow.

Animals↗