The formation of electron-donor-acceptor complexes for the detection of pesticides and their metabolites in conjunction with thin layer chromatography is discussed. The method is nondestructive, as the π-complex formation is reversible. Thus, the pesticide (or metabolite) may be recovered after separation from the complexing agent. The complexes studied may be transferred into a mass spectrometer sample tube and individual spectra of the pesticide and complexing agent may be obtained at different temperatures. The complexes are generally highly colored and this is an additional aid in distinguishing between closely related pesticides and metabolites. The absorption spectra of the chromatographed species on thin layer plates may be determined by in situ reflectance spectroscopy. The techniques discussed lack the sensitivity of methods such as esterase inhibition or fluorescence, but provide a high degree of selectivity for samples which may be separated at the microgram level or higher. The method of complexation and the instrumental techniques discussed could be useful to researchers involved in metabolic or structural studies.
<p>Functional integration of proteins with carbon-based nanomaterials such as nanotubes holds great promise in emerging electronic and optoelectronic applications. Control over protein attachment poses a major challenge for consistent and useful device fabrication, especially when utilizing single/few molecule properties. Here, we exploit genetically encoded phenyl azide photochemistry to define the direct covalent attachment of three different proteins, including the fluorescent protein GFP, to carbon nanotube side walls. Single molecule fluorescence revealed that on attachment to SWCNTs GFP’s fluorescence changed in terms of intensity and improved resistance to photobleaching; essentially GFP is fluorescent for much longer on attachment. The site of attachment proved important in terms of electronic impact on GFP function, with the attachment site furthest from the functional center having the larger effect on fluorescence. Our approach provides a versatile and general method for generating intimate protein-CNT hybrid bioconjugates. It can be potentially applied easily to any protein of choice; attachment position and thus interface characteristics with the CNT can easily be changed by simply placing the phenyl azide chemistry at different residues by gene mutagenesis. Thus, our approach will allow consistent construction and modulate functional coupling through changing the protein attachment position.</p>
We combined <i>in silico</i>modelling with fully genetically encoded strain promoted azide-alkyne cycloaddition, to construct bespoke protein dimers. Using fluorescent proteins GFP and Venus as models, homo and heterodimers were constructed that switched ON once assembled and displayed enhanced spectral properties. The determined molecular structure reveals long range polar bond networks involving amino acids and structured water molecules play a key role in activation and functional enhancement by directly linking the two functional centres. Single molecule analysis revealed the dimer is more resistant to photobleaching spending longer times in the ON state with only one CRO likely to be active at any one time. Thus, genetically encoded bioorthogonal chemistry can be used beyond simple passive linkage approaches to generate new and truly integrated protein complexes that form long range bonds networks, which have a profound effect on function and our understanding of fluorescent protein function.
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