Fluorescent Signal Transduction Research Articles

Design of a Membrane Fluorescent Sensor Based on Photo-Cross-Linked PEG Hydrogel

A polymeric hydrogel film was made photochemically for the fluorescent detection of copper ions. An eight-branched PEG macromer having a nitrocinnamate (NC) moiety as the pendant group was synthesized and found to form a gel upon exposure to 365-nm irradiation in the absence of any photoinitiators or catalysts. Peptide 1 (Dns-Gly-His-Lys(NC)) was synthesized with Fmoc solid-phase method and contained three essential parts: a dansyl fluorophore for fluorescent signal transduction, a metal binding site for selective recognition of copper ions from aqueous solution, and a NC group for interaction with PEG-NC macromer. The peptide was found to be efficiently immobilized within the hydrogel network during the photo-cross-linking process. The prepared PEG-NC/1 gel film is strongly fluorescent and can be quenched by the presence of copper ions. The quenching process was reversed by acid treatment and an excellent reversibility was obtained. The designed membrane sensor showed a superior selectivity toward coppe...

Fluorometric Detection of Ca2+ Based on an Induced Change in the Conformation of Sol−Gel Entrapped Parvalbumin

We report the development of a fluorometric detection strategy for Ca(2+) based on induced changes in the conformation of cod III parvalbumin entrapped within a sol-gel processed glass. The detection scheme utilizes a fluorescent allosteric signal transduction (FAST) strategy wherein conformational changes induced by Ca(2+) binding result in alterations in the intrinsic fluorescence from the single tryptophan residue at position 102. Intrinsic fluorescence was also used to examine chemically induced changes in protein structure to ascertain the effects of entrapment on the conformational motions and stability of the protein. Fluorescence analysis indicated that the behavior of the protein depended on the entrapment protocols used. The entrapped protein retained conformational flexibility similar to that observed in solution and remained accessible to analytes such as Ca(2+). Entrapment also caused improvements in protein stability against chemical denaturants. However, entrapment caused the apparent affinity constant for binding of Ca(2+) to decrease substantially with aging time. Even so, in optimum cases, fluorometric detection of Ca(2+) could be done over a 600 μM range with a limit of detection of 3 μM and with no interference from divalent ions such as Mg(2+), Sr(2+), or Cd(2+), indicating the viability of using sol-gel entrapped FAST proteins for the detection of Ca(2+).

Engineering Biosensors by Introducing Fluorescent Allosteric Signal Transducers: Construction of a Novel Glucose Sensor

The development of biosensors based on genetically engineered proteins offers many potential advantages to sensors that rely on natural proteins only. Here we present how protein engineering techniques can be used to integrate optical signal transduction functions directly into proteins by incorporating environmentally sensitive fluorescent groups that are allosterically linked to a ligand binding site, using a structure-based rational design approach. The location of potential fluorescent allosteric signal transduction (FAST) sites were predicted in the periplasmic Glucose Binding Protein of Escherichia coli. Single cysteine mutations were constructed at these locations, which allowed site-specific, covalent coupling of environmentally sensitive fluorophores. The fluorescence of these conjugates was shown to be hyperbolically dependent upon glucose concentration, indicating that the behavior of the fluorophore is allosterically coupled to the degree of occupancy at the glucose binding site, as predicted....