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Alan Van Orden

Publications and source records attributed to Alan Van Orden.

7 recordsLinked to original sources
PubMed2006

A three-state mechanism for DNA hairpin folding characterized by multiparameter fluorescence fluctuation spectroscopy.

The folding of a dye-quencher labeled DNA hairpin molecule was investigated using fluorescence autocorrelation and cross-correlation spectroscopy (FCS) and photon counting histogram analysis (PCH). The autocorrelation and cross-correlation measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset detection volumes, the relaxation time of the folding reaction, and the total concentration of DNA molecules participating in the reaction. The PCH measurements revealed the equilibrium distribution of DNA molecules in folded and unfolded conformations and the specific brightnesses of the fluorophore in each conformational state. These measurements were carried out over a range of NaCl concentrations, from those that favored the open form of the DNA hairpin to those that favored the closed form. DNA melting curves obtained from each sample were also analyzed for comparison. It was found that the reactant concentrations were depleted as the reaction progressed and that the equilibrium distributions measured by FCS and PCH deviated from those obtained from the melting curve analyses. These observations suggest a three-state mechanism for the DNA hairpin folding reaction that involves a stable intermediate form of the DNA hairpin. The reaction being probed by FCS and PCH is suggested to be a rapid equilibrium between open and intermediate conformations. Formation of the fully closed DNA hairpin is suggested to occur on a much longer time scale than the FCS and PCH measurement time. The closed form of the hairpin thus serves as a sink into which the reactants are depleted as the reaction progresses.

DNA↗
PubMed2006

Directing energy flow through quantum dots: towards nanoscale sensing.

Nanoscale sensors can be created when an expected energetic pathway is created and then that pathway is either initiated or disrupted by a specific binding event. Constructing the sensor on the nanoscale could lead to greater sensitivity and lower limits of detection. To this end, quantum dots (QDs) can be considered prime candidates for the active components. Relative to organic chromophores, QDs have tunable spectral properties, show less susceptibility to photobleaching, have similar brightness, and have been shown to display electro-optical properties. In this review, we discuss recent articles that incorporate QDs into directed energy flow systems, some with the goal of building new and more powerful sensors and others that could lead to more powerful sensors.

Journal Article↗
PubMed2005

Folding and unfolding kinetics of DNA hairpins in flowing solution by multiparameter fluorescence correlation spectroscopy.

Dynamic equilibrium between the folded and unfolded conformations of single stranded DNA hairpin molecules containing polythymine hairpin loops was investigated using simultaneous two-beam fluorescence cross-correlation spectroscopy and single beam autocorrelation spectroscopy. The hairpins were end-labeled with a fluorescent dye and a quencher, such that folding and unfolding of the DNA hairpin primary structure caused the dye fluorescence to fluctuate on the same characteristic time scale as the folding and unfolding reaction. These fluctuations were observed as the molecules flowed sequentially between two spatially offset, microscopic detection volumes. Cross-correlation analysis of fluorescence from the two detection volumes revealed the translational diffusion and flow properties of the hairpins, as well as the average molecular occupancy of the two volumes. Autocorrelation analysis of the fluorescence from the individual detection volumes revealed the kinetics of hairpin folding and unfolding, with the parameters relating to diffusion, flow, and molecular occupancy constrained to the values determined from the cross-correlation analysis. This allowed unambiguous characterization of the folding and unfolding kinetics, without the need to determine the hydrodynamic properties by analyzing a separate control sample. The analysis revealed nonexponential relaxation kinetics and DNA size-dependent folding times characteristic of dynamic heterogeneity in the DNA hairpin-forming mechanism.

Chemistry, Physical↗
PubMed2005

Single-feature inking and stamping: a versatile approach to molecular patterning.

A technique for micrometer-scale patterning of multiple functional biological molecules on surfaces is demonstrated. The technique is referred to as single-feature inking and stamping (SFINKS). It combines elements of dip-pen nanolithography and microcontact printing. "Inked" atomic force microscopy probes are used to ink individual features of an elastomer stamp. From a single stamp, we printed three different probe ssDNA with <10 mum resolution and showed that they specifically hybridize the complementary DNA labeled with different fluorophores. As a further demonstration of SFINKS' versatility, we patterned a silane onto a silicon wafer consisting of four subpatterns separated by >100 mum and composed of 2 mum lines. We discuss why patterns such as these are impractical with available techniques. Furthermore, we comment on the prospects for multiple stamping after a single inking.

Biomimetic Materials↗
PubMed2003

Two-beam fluorescence cross-correlation spectroscopy for simultaneous analysis of positive and negative ions in continuous-flow capillary electrophoresis.

Two-beam fluorescence cross-correlation spectroscopy was used for multicomponent electrophoretic analysis of positive and negative ions flowing continuously, and in opposite directions, through a polymer-coated electrophoresis capillary. Cross-correlation analysis of the fluorescence monitored from two spatially offset microscopic detection volumes revealed the magnitude and direction of the electrophoretic flow velocity of each analyte. This enabled resolution of a three-component mixture containing nanomolar concentrations of cationic rhodamine 6G and anionic 5-carboxytetramethylrhodamine (TAMRA) and TAMRA-labeled single-stranded DNA in aqueous buffer. The relative concentrations of each analyte, determined from the cross-correlation analysis, agreed with known values within experimental error. This technique is sensitive to molecular processes occurring on a millisecond time scale and will prove useful for monitoring rapid fluctuations in the magnitude and direction of the electrophoretic flow velocity caused by spontaneous chemical reactions or conformational transformations at thermodynamic equilibrium.

DNA, Single-Stranded↗