Unfolding of Helical Poly(L-Glutamic Acid) in N,N-Dimethylformamide Probed by Pyrene Excimer Fluorescence (PEF)

The denaturation undergone by α–helical poly(L-glutamic acid) (PLGA) in N,N-dimethylformamide upon addition of guanidine hydrochloride (GdHCl) was characterized by comparing the fluorescence of a series of PLGA constructs randomly labeled with the dye pyrene (Py-PLGA) to that of a series of Py-PDLGA samples prepared from a racemic mixture of D,L-glutamic acid. The process of pyrene excimer formation (PEF) was taken advantage of to probe changes in the conformation of α–helical Py-PLGA. Fluorescence Blob Model (FBM) analysis of the fluorescence decays of the Py-PLGA and Py-PDLGA constructs yielded the average number (<Nblob>) of glutamic acids located inside a blob, which represented the volume probed by an excited pyrenyl label. <Nblob> remained constant for randomly coiled Py-PDLGA but decreased from ~20 to ~10 glutamic acids for the Py-PLGA samples as GdHCl was added to the solution. The decrease in <Nblob> reflected the decrease in the local density of PLGA as the α–helix unraveled in solution. The changes in <Nblob> with GdHCl concentration was used to determine the change in Gibbs energy required to denature the PLGA α–helix in DMF. The relationship between <Nblob> and the local density of macromolecules can now be applied to characterize the conformation of macromolecules in solution.

Estimation of CYP3A4*1B single nucleotide polymorphism using target-assembled in-situ detection by synthetic DNA-mounted excimers

CYP3A4*1B is a single nucleotide polymorphism of CYP3A4 and is associated with prostate cancer which exhibits higher nifedipine oxidase activity in liver. This research provides details of the effects of structural variation and medium effects for the recently reported split-oligonucleotide (tandem) probe system for excimers-based fluorescence detection of DNA. In this approach the detection system is split at a molecular level into signal-silent components, which must be assembled correctly into a specific 3-dimensional structure to ensure close proximity of the excimer partners and the consequent excimer fluorescence emission on excitation. The model system consists of two 11-mer oligonucleotides, complementary to adjacent sites of a 22-mer DNA target. Each oligonucleotide probeis equipped with functions able to form an excimer on correct, contiguous hybridization. The extremely rigorous structural demands for excimer formation and emission required careful structural design of partners for excimer formation, which are here described. This study demonstrates that the excimer formed emitted at ~480 nm with alarge Stokes shift (~130 - 140 nm).

Synthesis of Di-Block Copolymers Poly (Propylene oxide)-block-Poly (9-(2,3-epoxypropyl) Carbazole) via Sequential Addition of Monomers in One Pot

Polypropylene oxide (PPO) and poly(9-(2,3-epoxypropyl) carbazole) (PEPK) di-block copolymers are prepared in one pot via sequential monomer addition by using i-PrONa/i-Bu3Al as an anionic catalytic system. An almost 100% monomer conversion is obtained, and the length of each block is controlled through the monomer/catalyst ratio used. Copolymer molecular weights are quite close to theoretical values calculated assuming the formation of one polymer chain per catalyst; therefore, it is hypothesized that the polymerization reaction proceeds with a living character. The synthesis appears to be particularly efficient and versatile. The calorimetric properties of copolymers obtained in this work are remarkable, since they show two distinct Tg values, corresponding to the PPO and PEPK blocks. The optical measurements of di-block copolymers show more analogous features than those of PEPK homopolymer. Copolymer solution emission spectra just exhibit isolated carbazole fluorescence, whereas in the solid state, film spectra show excimer fluorescence.

Computer vision vs. spectrofluorometer-assisted detection of common nitro-explosive components with bola-type PAH-based chemosensors

Simplified computer vision-assisted algorithm for the excimer fluorescence "turn-off" detection of nitro-analytes in aqueous media is described.

Supramolecular spectral/visual detection of urinary polyamines through synergetic/competitive complexation with γ-CD and CB[7]

A supramolecular strategy for detecting the concentration of polyamines has been established through competitive/synergetic complexation among polyamines, CB[7], γ-CD, and pyrene derivatives, which allows for convenient, rapid, and high throughput spectral/visual detection of the concentration of urinary polyamines based on the switching on/off of the pyrene excimer fluorescence.

Excimer–monomer fluorescence changes by supramolecular disassembly for protein sensing and quantification

A binding-induced supramolecular dissociation strategy is developed with the ratio of monomer and excimer fluorescence as the tool for protein sensing and quantification, exhibiting excellent selectivity.

Interior of Amylopectin and Nano-Sized Amylopectin Fragments Probed by Viscometry, Dynamic Light Scattering, and Pyrene Excimer Fluorescence

Nano-sized amylopectin fragments (NAFs), prepared by extrusion of waxy corn starch, were investigated by viscometry, dynamic light scattering (DLS), and pyrene excimer fluorescence (PEF). NAF57, with a hydrodynamic diameter of 57 nm, was treated with nitric acid to yield three degraded NAFs, which appeared to share the same interior and structural features as amylopectin based on their measured intrinsic viscosity and hydrodynamic diameter. This conclusion was further supported by comparing the efficiency of forming excimer between an excited and a ground-state pyrenyl label covalently attached to the NAFs (Py-NAFs) using their IE/IM ratio of the fluorescence intensity of the excimer (IE) to that of the monomer (IM). The overlapping trends obtained for all Py-NAFs and the pyrene-labeled amylopectin samples by plotting the IE/IM ratio as a function of pyrene content provided further evidence that the interior of NAFs and amylopectin shared the same structural features and contained a similar amount of free volume as predicted by the Solution-Cluster (Sol-CL) model. The presence of free volume was validated by adding linear poly(ethylene glycol) (PEG) chains that could not penetrate the interior of Py-NAFs, thus subjecting the Py-NAFs to increased osmotic pressure, which induced their compression and resulted in an increase in IE/IM.

Pyrene Excimer-Based Fluorescent Labeling of Cysteines Brought into Close Proximity by Protein Dynamics: ASEM-Induced Thiol-Ene Click Reaction for High Spatial Resolution CLEM

Fluorescence microscopy (FM) has revealed vital molecular mechanisms of life. Mainly, molecules labeled by fluorescent probes are imaged. However, the diversity of labeling probes and their functions remain limited. We synthesized a pyrene-based fluorescent probe targeting SH groups, which are important for protein folding and oxidative stress sensing in cells. The labeling achieved employs thiol-ene click reactions between the probes and SH groups and is triggered by irradiation by UV light or an electron beam. When two tagged pyrene groups were close enough to be excited as a dimer (excimer), they showed red-shifted fluorescence; theoretically, the proximity of two SH residues within ~30 Å can thus be monitored. Moreover, correlative light/electron microscopy (CLEM) was achieved using our atmospheric scanning electron microscope (ASEM); radicals formed in liquid by the electron beam caused the thiol-ene click reactions, and excimer fluorescence of the labeled proteins in cells and tissues was visualized by FM. Since the fluorescent labeling is induced by a narrow electron beam, high spatial resolution labeling is expected. The method can be widely applied to biological fields, for example, to study protein dynamics with or without cysteine mutagenesis, and to beam-induced micro-fabrication and the precise post-modification of materials.