PDMPO (2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole), has unique silica specific fluorescence and is used in biology to understand biosilicification. This 'silicaphilic' fluorescence is not well understood nor is the response to local environmental variables like solvent and pH. We investigated PDMPO in a range of environments: using UV-vis and fluorescence spectroscopy supported by computational data, (SPARC, molecular dynamics simulations, density functional theory calculations), dynamic light scattering and zeta potential measurements to understand the PDMPO-silica interaction. From absorption data, PDMPO exhibited a pKa of 4.20 for PDMPOH2(2+) to PDMPOH(+). Fluorescence emission measurements revealed large shifts in excited state pKa* values with different behaviour when bound to silica (pKa* of 10.4). PDMPO bound to silica particles is located in the Stern layer with the dye exhibiting pH dependent depolarising motion. In aqueous solution, PDMPO showed strong chromaticity with correlation between the maximum emission wavelength for PDMPOH(+)* and dielectric constant (4.8-80). Additional chromatic effects were attributed to changes in solvent accessible surface area. Chromatic effects were also observed for silica bound dye which allow its use as a direct probe of bulk pH over a range far in excess of what is possible for the dye alone (3-5.2). The unique combination of chromaticity and excited state dynamics allows PDMPO to monitor pH from 3 to 13 while also reporting on surface environment opening a new frontier in the quantitative understanding of (bio)silicification.
This paper describes novel adaptations of optically sectioned planar format assays to screen compounds for their affinities to materials surfaces. The novel platform, which we name Optical sectioned Indicator Displacement Assays (O-IDA), makes use of displaceable dyes in a format adaptable to high-throughput multi-well plate technologies. We describe two approaches; the first being where the dye exhibits fluorescence in both the surface bound and unbound state and the second, where fluorescence is lost upon displacement of the dye from the surface. Half maximal inhibitory concentration (IC50), binding affinity (Ki), and binding free energy (∆Gads) values can be extracted from the raw data. Representative biomolecules were tested for interactions with silica in aqueous environment and ZnO (0001)-Zn and (10-10) facets in a non-aqueous environment. We provide the first experimental values for both the binding of small molecules to silica and the facetdependent ZnO binding affinity of key amino acids associated with ZnO-specific oligopeptides. The specific data will be invaluable to those studying interactions at interfaces both experimentally and computationally. O-IDA provides a general framework for the high-throughput screening of molecules binding to materials surfaces, which has important applications in drug delivery, (bio-) catalysis, biosensing and biomaterials engineering.
Real-time analysis of macromolecular interactions and competitive binding of ligands to receptors on surfaces are not typically performed using fluorescence intensity methods due to background interference from solution fluorescence. Separation-free optically sectioned planar format assays (OSPFAs) with confocal detection remove this problem. We report OSPFAs for indicator displacement and kinetic assessment of binding. A commercial androgen receptor binding domain indicator displacement assay adapted as an OSPFA yielded an IC 50 of 6.5 nM for testosterone with Z 0 ¼ 0.77. These measured IC 50 and Z 0 values are in ranges suitable for drug screening applications with Z 0 > 0.5 indicating good to excellent screenability. An OSPFA was applied to study the rate of antibody binding to a sandwich immunoassay on a planar surface. Langmuir fits provided forward rate constants in the range 2 Â 10 3 M À1 s À1 to 6 Â 10 4 M À1 s À1 and reverse constants 1 Â 10 À4 s À1 to 4 Â 10 À3 s À1 which cover a useful range for characterising probe-target interactions. This work demonstrates the suitability of OSPFAs for investigating kinetics and binding interactions using fluorescence under wash-free conditions. OSPFAs are an alternative to other separation-free methods such as acoustic, surface plasmon resonance, ellipsometry, fluorescence polarisation and other related methods. OSPFAs should make practical dynamic binding studies for small and large molecules including cases where the reaction under investigation results in no appreciable mass change on a surface.
The lipid crystals are one of the important hallmarks of advanced atherosclerotic plaques. However, the detailed assessment of lipid crystals including their chemical compositions and morphological information at the tissue level are little known due to the lack of appropriate imaging techniques. Here, we developed a combinational method to characterize the morphology of lipid crystals precisely and concomitantly to identify their chemical compositions based on coherent anti-stokes raman scattering (CARS) spectroscopy and
We describe a surface charge imaging method for heterogeneous biosilicas based on relationships between zeta (ζ) potential, feature size of nanoparticles, and PDMPO fluorescence and apply it to silicified structures...
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