Biomedical subjects

Jennifer S Shumaker-Parry

Publications and source records attributed to Jennifer S Shumaker-Parry.

4 recordsLinked to original sources

PubMed2004

A streptavidin linker layer that functions after drying.

The ability of streptavidin (SA) to simultaneously bind four biotins is often used in linker layers, where a biotinylated molecule is linked to a biotin-functionalized surface via SA. For biosensor and array applications, it is desirable that the SA linker layer be stable to drying and rehydration. In this study it was observed that a significant decrease in binding capacity of a SA layer occurred when that layer was dried. For this study a SA linker layer was constructed by binding SA to a biotin-containing alkylthiolate monolayer (BAT/OEG) self-assembled onto gold. Its stability after drying was investigated using surface plasmon resonance (SPR). Approximately a quarter of the SA layer was removed from the BAT/OEG surface upon drying and rehydration, suggesting disruption of SA-biotin binding when dry. This resulted in the dried SA layer losing approximately 40% of its biotinylated ferritin (BF) binding capacity. Coating the layer with trehalose before drying was found to inhibit the loss of SA from the BAT/OEG surface. SPR showed that the trehalose-protected SA linker layer retained approximately 91% of its original BF binding capacity after drying and rehydration. Atomic force microscopy, which was used to image individual surface-bound SA and BF molecules, qualitatively confirmed these observations.

Biotin↗

PubMed2004

Parallel, quantitative measurement of protein binding to a 120-element double-stranded DNA array in real time using surface plasmon resonance microscopy.

Quantitative, real-time measurement of kinetics of sequence-specific binding of DNA-binding proteins to double-stranded DNA (dsDNA) immobilized in a 10 x 12 array on a planar gold surface is demonstrated using surface plasmon resonance (SPR) microscopy. This binding of the yeast transcription factor Gal4 to a 120-spot dsDNA array made with alternating 200-microm spots of its dsDNA operator sequence and an unrelated DNA sequence proves that this method could be used to simultaneously monitor the kinetics of binding of proteins to 120 different dsDNA sequences with a sensitivity to approximately 0.5 pg (<2 x 10(7) molecules) of bound protein in each array spot at a time resolution of 1 s. The method is label free and also allows absolute quantitative determination of the binding stoichiometry (i.e., the number of proteins bound per dsDNA) at each time. The dsDNA array was fabricated using a robotic microspotting system to deliver nanoliter droplets of biotinylated dsDNA solutions onto a streptavidin linker layer immobilized with biotinylated alkylthiols on a thin gold film. Simultaneous monitoring of binding to the many array elements allows the use of reference spots (i.e., array elements with unrelated dsDNA sequences) to correct the signal for nonspecific protein-DNA binding and changes in bulk refractive index of the solutions in the SPR microscope's flow cell. This allows high-throughput analyses of the kinetics and equilibrium of protein-dsDNA binding.

Animals↗

PubMed2004

Quantitative methods for spatially resolved adsorption/desorption measurements in real time by surface plasmon resonance microscopy.

A simple method for converting local reflectivity changes measured in surface plasmon resonance (SPR) microscopy to effective adlayer thicknesses and absolute surface coverages of adsorbed species is presented. For a range of high-contrast angles near the SPR resonance where the local metal surface's reflectivity changes linearly with angle, the change in reflectivity at fixed angle is proportional to the change in effective refractive index (eta(eff)) near the surface. This change in eta(eff) can be converted to absolute adsorbate coverage using methods developed for quantitative SPR spectroscopy. A measurement of the change in reflectivity due to changes in refractive index of bulk solutions, i.e., percent reflectivity change per refractive index unit (RIU), is the only calibration required. Application of this method is demonstrated for protein adsorption onto protein/DNA arrays on gold from aqueous solution using an SPR microscope operating at 633 nm. A detection limit of 0.072% change in absolute reflectivity is found for simultaneous measurements of all 200 microm x 200 microm areas within the 24-mm(2) light beam with 1-s time averaging. This corresponds to a change in effective refractive index of 1.8 x 10(-5) and a detection limit for protein adsorption of 1.2 ng/cm(2) (approximately 0.5 pg in a 200-microm spot). The linear dynamic range is Deltaeta(eff) = approximately 0.011 RIU or approximately 720 ng/cm(2) of adsorbed protein. Using a nearby spot as a reference channel, one can correct for instrumental drift and changes in refractive index of the solutions in the flow cell.

Adsorption↗

PubMed2004

Microspotting streptavidin and double-stranded DNA arrays on gold for high-throughput studies of protein-DNA interactions by surface plasmon resonance microscopy.

We present two strategies for microspotting 10 x 12 arrays of double-stranded DNAs (dsDNAs) onto a gold-coated glass slide for high-throughput studies of protein-DNA interactions by surface plasmon resonance (SPR) microscopy. Both methods use streptavidin (SA) as a linker layer between a biotin-containing mixed self-assembled monolayer (SAM) and biotinylated dsDNAs to produce arrays with high packing density. The primary mixed SAM is produced from biotin- and oligo(ethylene glycol)-terminated thiols bonded as thiolates onto the gold surface. In the first method, a robotic microspotter is used to deliver nanoliter droplets of dsDNA solution onto a uniform layer of this SA ( approximately 2 x 10(12) SA/cm(2)). SPR microscopy shows a density of (5-6) x 10(11) dsDNA/cm(2) (0.2-0.3 dsDNA/SA) in the array elements. The second method uses instead a microspotted array of this SA linker layer, onto which the microspots of dsDNA are added with spatial registry. SPR microscopy before addition of the dsDNA shows a SA coverage of 2 x 10(12) SA/cm(2) within the spots and a dsDNA density of 8.5 +/- 3.5 x 10(11) dsDNA/cm(2) (0.3-0.7 dsDNA/SA, depending on the length of dsDNA) after dsDNA spotting. We demonstrate the ability to simultaneously monitor protein binding with the SPR microscope in many 200-microm spots with 1-s time resolution and sensitivity to <1 pg of protein.

Adsorption↗