Pyranine Molecules Research Articles

Front Cover: Highly Sensitive Fluorescent pH Microsensors Based on the Ratiometric Dye Pyranine Immobilized on Silica Microparticles (Chem. Eur. J. 53/2021)

Optical fluorescent pH sensors based on positively charged silica microparticles containing anionic pyranine molecules were developed by using a modified Stöber method with a gradual immobilization of ratiometrically pH-sensing pyranine molecules during the growth of silica seed particles and an overall positive charge in a one-step reaction. The overly pH-sensitive nature of pyranine is preserved and long-term stability is ensured by silica shell coating. These cytocompatible ratiometric pH sensors can be used for pH sensing and pH tracking applications where each microparticle can be observed individually due to resolvable size. More information can be found in the Communication by A. Chandra, L. L. del Mercato et al. (DOI: 10.1002/chem.202101568).

Highly Sensitive Fluorescent pH Microsensors Based on the Ratiometric Dye Pyranine Immobilized on Silica Microparticles.

Invited for the cover of this issue are Anil Chandra, Loretta L. del Mercato and co-workers at the Institute of Nanotechnology of National Research Council and the University of Salento. The image depicts how negatively charged pH-sensitive pyranine (HPTS) molecules were successfully immobilized on silica microparticles (SMPs) without compromising the molecules' pH sensitivity. These resulting sensors can be used to measure pH in vitro and in vivo due to the cytocompatibility of HPTS molecules and SMPs. Read the full text of the article at 10.1002/chem.202101568.

Excited state proton transfer processes of pyranine studied by femtosecond stimulated Raman spectroscopy

As one of the most fundamental processes, proton transfer reaction plays an important role in chemical and biological process, and to reveal the choreography of the proton motion intra- and intermolecularly, a spectroscopic technique capable of capturing molecular structural dynamics of excited-state proton transfer motions on an intrinsic time scale is needed. In this study, We utilize wavelength-tunable femtosecond stimulated Raman spectroscopy with a time resolution of ~100 fs, spectral resolution of 15 cm<sup>–1</sup> and spectral range of 400 cm<sup>–1</sup>—1800 cm<sup>–1</sup>, combined with traditional transient absorption spectroscopywith a time window between 0 and 600 ps to simultaneously achieve reaction dynamics for transient reactant and product of the photoacid pyranine (8-hydroxypyrene1, 3, 6-trisulfonic acid, HPTS) molecules undergoing excited-state proton transfer reaction in complex with water and acetate molecules. Marker bands attributed to the deprotonated form of HPTS in a frequency range from 400 cm<sup>–1</sup> to 1700 cm<sup>–1</sup> are obtained under the excitation of 400 nm laser pulses. The marker band at 1516 cm<sup>–1</sup>, which is assigned to phenolic ring carbon carbon double band stretching accompanied with carbon hydrogen in-plane rocking motions, exhibits complex rise and decay dynamics. The simultaneously observed excited-state Raman mode at 920 cm<sup>–1</sup> which is assigned to the excited carbon-carbon single bond stretch mode in the protonated acetic acid root molecule, helps us to clearly resolve the reaction rates of excited-state proton transfer. Based on the multi-exponential fitting results, the dynamics of excited-state Raman mode at 920 cm<sup>–1</sup> exhibits bi-exponential processes with time constants of ~470 fs and ~3 ps. The ultrafast time component indicates that the excited-state proton transfer originates from an HPTS-acetate complex, indicating that part of the ground-state HPTS molecules are in the “tight” hydrogen bonding configuration that can quickly shift the excited-state proton charge toward the acetate acceptor molecule through a direct hydrogen bond. The second slower time component implies a significant subpopulation of HPTS in the ground state, i.e. hydrogen bonds to an acetate ion via an intervening water molecule, and upon photo excitation, the proton transfers to the water solvent before proton is picked up by the acetate ion.

CNTs based Water soluble fluorescent sensor for selective detection of Fe3+ ion

Herein, a fluorescence sensor based on the fluorescence quenching phenomena was developed by the functionalization of Pyranine molecules over the surface of carbon nanotubes (Pry-CNTs) for quick, selective and sensitive detection of Fe3+ ion. The change in the emission along with the Fluorescence Quenching response was found linearly comparative to the Fe3+ ion concentration in the range of 0.1−10 μM, with the limit of detection of 0.2 μM. The Fluorescence study proved the coordination interaction between Fe3+ ion and Pry-CNTs lead to the recognition process. The developed fluorescence sensor is the simple and selective link for the determination of Fe3+ ion. The recovery of this method in the determination of Fe3+ ion in real water samples was found to be 99.5–100 %. This fluorescence sensor (Pyr-CNTs) can be used as a sensor for Fe3+ ion and promote for many applications in the environmental, chemical and biological systems.

Gelation of PAAm-PVP composites: A fluorescence study

Hybrid hydrogels are a new class of composite materials. Polyacrylamide (PAAm) hydrogels are mainly produced by free radical crosslinking copolymerization (FCC) of AAm in the presence of N, N′-methylene bis (acrylamide) (BIS) as the crosslinker. Pyranine doped PAAm-poly (N-vinyl pyrrolidone) (PVP) composite were prepared with different amounts of PVP varying in the range between 0.0015 and 0.1 gr. It was observed that pyranine molecules as a fluoroprobe bind to AAm and PVP chains upon the initiation of the polymerization, causing the fluorescence spectra of the bonded pyranines shift to the shorter wavelengths. The sol–gel phase transition and its universality were monitored and tested as a function of PVP contents. Observations around the critical point show that the gel fraction exponent, β, agreed with the percolation result for below 0.025 gr PVP contents. However, classical result was observed above 0.0125 gr PVP content.

Magnetic hydrogel with high coercivity

This study investigates the synthesis and characterization of polyacrylamide (PAAm) hydrogels containing magnetic BaFe12O19 nanoparticles. Structural, electrical, and magnetic characterization of the gels have been performed with X-ray powder diffractometry, scanning electron microscopy, DC conductivity, magnetization and fluorescence spectroscopy techniques. The preparation and characterization of polyacrylamide (PAAm) hydrogels that contain 5 and 10mg BaFe12O19 (16 and 21nm diameter) nanoparticles are described herein. It is seen from the fluorescence spectra that, nanoparticles surrounded to pyranine molecules so that some of pyranine molecules could not bound to the polymer strands. Electrical measurements show that presence of nanoparticles make the gel more homogeneous. Magnetization measurements reveal that hydrogels have hard magnetic properties with quite high coercivity of 4.2kOe, which does not change with swelling. This feature makes these gels useful in applications as wastewater treatment if they are magnetized before use.

Fluorescence and magnetic properties of hydrogels containing Fe3O4 nanoparticles

In this study, Fe3O4 (magnetite) nanoparticles were synthesized by in situ in polyacrylamide (PAAm) gels. Structural and magnetic properties of magnetite nanoparticles were investigated by X-Ray diffractometry (XRD), transmission electron microscopy (TEM) and vibrating sample magnetometry (VSM) methods. Interaction of pyranine molecules with Fe3O4 nanoparticles was investigated by fluorescence spectroscopy measurements in which pyranine (POH) molecules were used as a flouroprobe. It was observed that magnetite nanoparticles have single domain structure with average grain size of 12nm which was also supported by magnetization measurements. M–H hysteresis curves revealed the superparamagnetic nature of magnetite nanoparticles synthesized in PAAm gels. Fluorescence measurements depicted that there is an interaction between POH and magnetite nanoparticles which was deduced from the presence of two new peaks at 380nm and 405nm in emission spectrum. Besides, it was observed that POH molecules could not diffuse into the gel when it consists of magnetite nanoparticles which also make the gel more homogeneous. As swelling ratio increases from 1 to 13, magnetization of the gel does not change which reveals that magnetic nanoparticles do not diffuse out of the gel during water-intake. This feature makes the gel suitable for applications as waste water treatments.

多孔質陽極酸化アルミナに埋め込んだピラニン分子の吸収および蛍光特性

多孔質陽極酸化アルミナに埋め込んだピラニン分子の吸収および蛍光特性

Magnetic and spectroscopic properties of Polyacrylamide-CoFe2O4 magnetic hydrogel

This study investigates synthesis and characterization of polyacrylamide (PAAm) hydrogels containing ferromagnetic CoFe2O4 nanoparticles. Structural, electrical, and magnetic characterization of the gels have been performed with X-ray powder diffractometry, Scanning electron microscopy, DC conductivity, magnetization and fluorescence spectroscopy techniques. Fluorescence and electrical measurements show that nanoparticles have trapped in the gel so they cannot move through the gel even if the gel is swollen and the voltage is applied. Pyranine molecules diffuse easily through the gel due to the presence of ferromagnetic nanoparticles. As number of diffused pyranine molecules increases current densities of the magnetic hydrogel increase. Magnetization measurements reveal that CoFe2O4 nanoparticles do not diffuse out of the gel during swelling. As a result, total magnetization of the gel do not change as volume of the gel increases.

Monitoring the gelation of polyacrylamide–sodium alginate composite by fluorescence technique

Polyacrylamide (PAAm)–sodium alginate (SA) composite was prepared with different amounts of SA varying in the range between 0.06% and 2% (w/v). The PAAm–SA composite was characterized by the steady-state fluorescence technique. Pyranine was added as a fluoroprobe for monitoring the polymerization. It was observed that pyranine molecules bind to AAm and SA chains upon the initiation of the polymerization. Thus, the fluorescence spectra of the bonded pyranines shift to the shorter wavelengths. Fluorescence spectra from the bonded pyranines allowed us to monitor the sol–gel phase transition, and to test the universality of the sol–gel transition as a function of SA contents. Observations around the critical point show that the gel fraction exponent, β, and the weight average degree of polymerization exponent, γ, agreed with the percolation result for (<0.25% (w/v)) SA contents. However, classical results were produced at (<2% (w/v)) SA contents.

The Role of Pyranine in Characterization of PAAm-κC Composites by Using Fluorescence Technique

Polyacrylamide (PAAm) doped by κ-carrageenan (κC) gels were prepared with various amounts of κC varying in the range between 0 wt.% and 3 wt.%. Steady-state fluorescence (SSF) technique was employed for studying sol-gel transition and swelling of PAAm-κC composite gels which were prepared by free-radical crosslinking copolymerization. Pyranine was introduced as a fluorescence probe. Pyranine molecules start to bind to acrylamide polymer chains upon the initiation of the polymerization, thus the spectra of the bonded pyranines shift to the shorter wavelengths. Fluorescence spectra from the bonded pyranines allow one to monitor the sol-gel transition and to test the universality of the sol-gel transition as a function of some kinetic parameters like polymer concentration. Observations around the gel point, t ( c ) for PAAm-κC composite gels showed that the gel fraction exponent β obeyed the percolation result for low κC (<2.0 wt. %) however classical results were produced at higher κC (>2.0 wt.%). On the other hand, fluorescence intensity of pyranine was measured during in situ swelling process at various amounts of κC and it was observed that fluorescence intensity values decreased as swelling is proceeded. Li-Tanaka equation was used to determine the swelling time constants, τ and cooperative diffusion coefficients, D.

Photophysical Changes of Pyranine Induced by Surfactants: Evidence of Premicellar Aggregates

We studied photophysical changes of pyranine in different surfactant environments using spectrophotometry, steady-state fluorescence, and time-resolved fluorescence lifetime. The effect of surfactants on such properties varied as a function of the surfactant charge. Whereas anionic surfactants did not show any kind of interaction, the nonionic surfactant Triton X-100 produced spectral changes in the dye, as a consequence of the shift of equilibrium between its excited species. In the case of fluorescence, these changes allowed the critical micellar concentration of the surfactant to be determined. However, the most important features were obtained from the interaction with cationic surfactants of the n-alkyl trimethylammonium bromides. Such interactions enabled the formation of premicellar aggregates to be determined. In addition, three or four critical concentrations could be defined, which were dependent on the length of the hydrocarbon chain. One of these was the critical micellar concentration of the surfactant. The remaining two or three were in the very dilute concentration domain from which several types of premicellar aggregates are formed. However, the interaction with dodecyltrimethylammonium bromide at submicellar concentrations evidenced a quenching effect of a different nature (either static or combined), depending on the surfactant concentration. By deconvoluting the overall pyranine fluorescence emission spectrum into a sum of overlapping Lorentzian-Gaussian functions, the principal microenvironments of the pyranine molecules can be ascertained. Fluorescence data are consistent with the location of pyranine in a variety of sites, including partitioning that may be influenced by electrostatic and pi-cation interactions in aqueous micelles.

Steady state fluorescence technique for studying phase transitions in PAAm–PNIPA mixture

The steady state fluorescence (SSF) technique was used to study the sol-gel phase transition during free radical crosslinking copolymerisation of various amounts of acrylamide (AAm) and N-isopropylacrylamide (NIPA) mixtures. N,N′-methylenebis (acrylamide) (BIS) and ammonium persulfate (APS) were used as crosslinker and an initiator, respectively. Pyranine (8-hydroxypyrene-1, 3, 6-trisulfonic acid, trisodium salt, HPTS) was added as a fluoroprobe for monitoring the polymerisation. It was observed that pyranine molecules bind to AAm and NIPA chains upon the initiation of the polymerisation, thus the fluorescence spectra of the bonded pyranines shift to the shorter wavelengths. Fluorescence spectra from the bonded pyranines allowed us to monitor the sol-gel phase transition, without disturbing the system mechanically, and to test the universality of the sol-gel transition as a function of polymer concentration ratios. Observations around the gel point of PAAm–PNIPA mixtures show that the gel fraction exponent β obeyed the percolation result.

Change in the Orientation and Packing upon Adsorption of Pyranine Molecules onto Cationic Langmuir Monolayers and Langmuir–Blodgett Films

Abstract Differrent adsorption behaviors and the orientation of pyranine molecules were investigated for the cationic Langmuir monolayers by using Brewster angle microscopy (BAM), two-dimensional (2D) phase behavior with elasticitic property of the monolayers and in situ visible spectroscopy. The structural change in the cationic Langmuir–Blodgett (LB) films before and after adsorbing pyranine molecules was found by using small angle and in-plane XRD. The excited-state proton-transfer (ESPT) between octadecylamine (ODA) and pyranine adsorbed in the films was investigated using time-resolved fluorescence spectroscopy together with consideration of the film structures.

Study on critical behaviour in N -isopropyl acrylamide gels by using fluorescence technique

The steady state fluorescence (SSF) technique was used to study the sol-gel transition for the solution free radical crosslinking copolymerization of N-isopropyl acrylamide (NIPA), with N,N′-methylenebis (acrylamide) (BIS) as crosslinker in the presence of ammonium persulfate (APS) as an initiator. Pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt, HPTS) was used as a floroprobe for monitoring the polymerization. Pyranine molecules start to bind to NIPA polymer chains upon the initiation of the polymerization, thus the spectra of the bonded pyranines shift to the shorter wavelengths. The gel fraction exponent β and the weight average degree of polymerization exponent γ′ agree best with the mean-field (Flory-Stockmayer) results near the gel point for various crosslinker contents.

Universal Behaviour of Gel Formation from Acrylamide-Carrageenan Mixture Around the Gel Point: A Fluorescence Study

The steady state fluorescence (SSF) technique was used to study the sol-gel transition, for the solution free radical crosslinking copolymerization of acrylamide (AAm) with various carrageenan content. N, N′- methylenebis (acrylamide) (BIS) and ammonium persulfate (APS) are used as crosslinker and an initiator, respectively. Pyranine (8-hydroxypyrene-1, 3,6-trisulfonic acid, trisodium salt, HPTS) was added as a floroprobe for monitoring the polymerization. Pyranine molecules start to bind to acrylamide polymer chains upon the initiation of the polymerization; thus, the spectra of the bonded pyranines shift to the shorter wavelengths. Fluorescence spectra from the bonded pyranines allows one to monitor the sol-gel transition, without disturbing the system mechanically, and to test the universality of the sol-gel transition as a function of some kinetic parameters like polymer concentration. Observations around the critical point show that the gel fraction exponent β obeyed the percolation result for low carrageenan concentrations (<2.0%) however classical results were produced at higher carrageenan concentration (>2.0%).

Fabrication of Microarrays on Fused Silica Plates Using the Laser-Induced Backside Wet Etching Method

A novel approach in the fabrication of microarrays of dye and protein on fused silica plates using the laser-induced backside wet etching (LIBWE) technique is described. The surface of fused silica plates was initially precoated using trimethoxysilane self-assembled monolayers (SAMs) and then etched using the LIBWE method to obtain the desired microstructures on the plate surface. Using this technique, the SAMs on the nonirradiated areas were able to survive the LIBWE process and were used as templates for the subsequent deposition of dye molecules or proteins via chemical bonding or physical adsorption. In the case of fused silica plates precoated with fluorinated SAMs, the LIBWE method is used to remove the SAMs to expose the etched silica surfaces, on which a thin layer of pyranine molecules can be site-selectively deposited using an aqueous solution of pyranine. In another application, an ethanol solution of rhodamine 6G was preferentially deposited onto the nonirradiated areas. In yet another application, bovine serum albumin was preferentially deposited onto the laser-irradiated areas; in this case, the fused silica plates were precoated with poly(ethylene oxide) SAMs. Interestingly, when an aqueous suspension of polystyrene (PS) microbeads was cast onto the fused silica precoated with the fluorinated SAMs, hexagonally close-packed PS microbeads were deposited into the etched cavities. Depositions of the dye, protein, and microbeads were confirmed by visualization using a fluorescence microscope and scanning electron microscope.

Direct test of the critical exponents at the sol-gel transition.

The steady state fluorescence technique was used to study the sol-gel transition for the solution-free radical cross-linking polymerization of acrylamide (AAm), with N,N'-methylenebis (acrylamide) as cross linker in the presence of ammonium persulfate as an initiator. Pyranine (8-hydroxypyrene-1, 3,6-trisulfonic acid, trisodium salt) is used as a fluoroprobe for monitoring the polymerization. Pyranine molecules start to bind to acrylamide polymer chains upon the initiation of the polymerization, thus the spectra of the bonded pyranines shift to the shorter wavelengths. Fluorescence spectra from the bonded pyranines allows one to monitor the sol-gel transition, without disturbing the system mechanically, and to test the universality of the sol-gel transition as a function of some kinetic parameters such as polymer concentration, cross-linker concentration, and temperature. Observations around the critical point show that there are three regimes for AAm concentration in which the exponents differ drastically. The gel fraction exponent beta and the weight average degree of polymerization exponent gamma agree best with the static percolation results for higher acrylamide concentrations above 1M, but they cross over from percolation to mean-field (Flory-Stockmayer) values when the AAm concentration is lower than 2M. For very low polymer concentrations, below which the system can not form the gel, the exponents differ considerably from both the percolation and the mean-field values.

The most probable trajectory for ion flux through large-pore channel

Large-pore channels are β-barrel proteins that transverse the bacterial outer membrane, forming partially selective pores that are ∼50 Å long and ∼30 Å wide. Through these proteins, whose structures were determined up to 2 Å resolution, small solutes permeate into the periplasmic space of the bacteria. In the present study, we investigated the propagation of ions inside the channel space using a combination of sub-nanosecond time-resolved fluorescence measurements and numeric reconstruction of propagation dynamics in order to characterize the properties of the diffusion space inside the channel. The experimental system consisted of the insertion of a single pyranine molecule at the center of the ion-conducting large-pore channel protein (PhoE) and synchronous dissociation of the pyranine molecules by a laser flash. Time-resolved fluorescence monitored the recombination of the excited pyranine anion with the released proton. The electrostatic potential inside the ion-conducting channel was calculated by the DelPhi program, using as input the three-dimensional structure of the protein. The propagation of the proton in the diffusion space was carried out stepwise; first the most probable trajectory was defined, thus reducing the system into an 1D diffusion problem. Once the trajectory was determined, the variation of the entropy along the propagation pathway was calculated. The gradients of the electrostatic potential and the entropy were combined into a transition probability term that determined the probability of a proton to propagate along the trajectory. The propagation algorithm was used sequentially, with the intra-cavity dielectric constant as an adjustable parameter, until the calculated dynamics were super-positioned over the experimental signal. The dielectric constant of the aqueous space inside the channel has a value of ε intra-cavity=50±5. The validity of the procedure was tested by applying the same algorithms for calculating the passage time of positive and negative charges through five proteins of the large-pore channel family. The calculated first passage times were within ±50% of the experimentally determined mean passage times reported in the literature.

Effects of probe-amphiphile interaction on pyranine proton transfer reactions in lecithin vesicles

Photoacids and photobases are powerful tools for the discrimination, in real time, between the preferential pathways in H +/OH − transfer processes in biological and biomimetic systems which can determine the origins of crucial biochemical processes. In this study, the binding of 8-hydroxy-1,3,6-pyrenetrisulphonate sodium salt (pyranine) to phosphatidylcholine (lecithin) vesicles was investigated (by time-resolved and steady state fluorometry and laser-induced pH jump measurements) as a function of the probe and electrolyte concentration to determine how these parameters affect the properties of the photoacid. The ratio ( R) between the fluorescence intensities at 510 nm (dissociated form) and 440 nm (undissociated form) can be related to the excited state degree of dissociation ( D (%)). The binding of the probe to the internal leaflet of lecithin small unilamellar vesicles (SUVs) is larger than that to the external leaflet. The addition of salt up to 2 M does not prevent binding even at low probe concentrations. Time-resolved emission decay measurements also show the probe-lipid interaction. The lifetime of the undissociated form is equivalent to that in alcoholic solutions (approximately 3.8 ns). The pyranine ground state re-protonation rate constant ( k obs) is dependent on the probe content per vesicle. With 550 pyranine molecules per vesicle, k obs is constant within a large range of salt concentration. In this situation, an alternative route is proposed (compared with direct PO −/H + recombination), in which proton transfer between neighbouring species maintains an unchanging recombination rate. The data presented show that careful analysis of pyranine H + transfer data is necessary for biomimetic and biological or reconstituted systems.