Nano-Conjugate Fluorescence Probe for the Discrimination of Phosphate and Pyrophosphate

We describe a pyrophosphate (PPi) probe that is based on a fluorescent dicarboxylate-substituted poly(para-phenyleneethynylene) (PPE) and 10 nm cobalt–iron spinel nanoparticles (NPs) in aqueous media. The spinel NPs efficiently quench the fluorescence of the PPE at a concentration of 20–30 pmol. Addition of phosphate anions to the PPE–NP construct displaces the quenched PPE to give rise to a fluorescent response; we found that PPi and phosphate (Pi) have significantly different binding affinities for the self-assembled materials. We can discern \u3e40 nM PPi in the presence of 0.1 mM Pi at pH 7, which suggests that these assemblies may be useful in bio-analytical applications. This displacement assay was used to effectively determine the ability of pyrophosphatase to hydrolyze PPi to Pi

Luminescent vesicular receptors for the recognition of biologically important phosphate species

The anion binding ability of bis-zinc cyclen complexes in buffered aqueous solution was investigated using indicator displacement assays (IDA) as well as luminescent labelled complexes. A high affinity to phosphate anions, such as UTP or pyrophosphate was observed in IDA while there was no observable binding of other anions. The binding affinity, and as a result the selectivity, between different phosphate anions correlates with their overall negative charge and steric demand. Complexes bearing luminescent labels did not respond to the presence of phosphate anions in homogeneous solution, but did if embedded as amphiphiles in small unilamellar vesicle (SUV) membranes. The scope of possible anionic analytes was extended to phosphorylated protein surfaces by using such metal complex-functionalized vesicles bearing oligoethylene glycol residues in an optimized amount on their surface to suppress non-specific interactions. Under physiological conditions these surface-modified vesicles show a selective response and nanomolar affinity for alpha-S1-Casein, which is multiple phosphorylated, while not responding to the corresponding dephosphorylated Casein or BSA. The vesicular luminescent metal complexes do not currently reach the sensitivity and selectivity of reported enzymatic assays or some chemosensors for phosphate anions, but they present a novel type of artificial receptor for molecular recognition. Membrane-embedding of multiple, different receptors and their possible structuring on the vesicular surface is expected to improve affinities and selectivities and may allow the design of artificial antibodies