Kaoru Iwai

Development of fluorescent microgel thermometers based on thermo-responsive polymers and their modulation of sensitivity range

Fluorescent molecular thermometers based on thermo-responsive linear polymer molecules such as poly(N-isopropylacrylamide) (PNIPAM) labelled with a polarity-responsive fluorescent molecule benzofurazan (BD) are the most sensitive known. Thermo-responsive PNIPAM and some related polymer microgel particles labelled with BD by emulsion polymerization have been prepared and their fluorescence properties in water as fluorescent thermometers studied. All the cross-linked polymer microgel particles dispersed in water fluoresce strongly as soon as each threshold temperature is exceeded. The nine kinds of microgel dispersion developed in this work thoroughly cover the sensitivity range from 18 to 47 °C. They are not only more sensitive than the previous fluorescent molecular thermometers based on other principles but also highly reproducible in their behaviour.

Multiplexing Sensory Molecules Map Protons Near Micellar Membranes

Fluorescent sensors have great potential to operate as molecular-level devices in nanospaces. Generally, a fluorescent sensor monitors a single parameter of its local environment, such as ion concentration. More functionalized systems which operate according to similar principles are molecular logic gates. These gates respond to multiple parameters simultaneously according to defined Boolean transformations. There are also a few examples of molecular sensors which respond to multiple parameters, each by a different analytical technique. Herein we demonstrate a new multiplexing fluorescent sensor which simultaneously monitors multiple parameters (local proton concentration and polarity in this instance) by multiple emission properties (intensity and wavelength, respectively). As the polarity of spherical micelles in water is expected to change largely monotonically along a radial coordinate, polarity data translate into positions. We can thus obtain local proton densities at various positions by scattering a series of multiplexing sensors widely over the aqueous micellar field. Therefore a nanoscaled mapping of proton concentration emerges for this simple membrane system. Proton concentration gradients are responsible for the subject of bioenergetics. Multiplexing sensors also correspond to nanoscale versions of robotic vehicles which go to humanly inaccessible spaces, map local properties and send information back to us. Scheme 1 shows the structures of the fluorescent multiplexing sensors 1–18 used in this study. These sensors consist of a polarity-sensitive fluorophore (blue), a proton receptor (orange), position tuners (red), and a spacer (green). The sensors function as follows: 1) The local proton concentration is examined by a DpKa value (pKa in micellar solution–pKa in water) of a conjugate acid of the receptor amine. This DpKa value is affected by electrostatic potential and dielectric constant at the sensor location but is independent of intrinsic acidity/basicity of the sensor. If local effective proton concentration is higher than that of bulk water, a positive DpKa value is obtained. [9] As our sensors possess a fluorescence “off–on” switching system by controlling photoinduced electron transfer processes with a fluorophore–spacer–receptor format, the DpKa values can be determined from fluorescence intensity, with pH profiles arising from titrations. 2) The local polarity is estimated from the emission wavelength of the polarity-sensitive fluorophore, 4-sulfamoyl-7aminobenzofurazan, as its emission wavelength is strongly red-shifted with increasing environmental polarity and is smoothly related to the dielectric constant e of the solvent. Thus, the relationship between the emission wavelength and the e value is obtained beforehand for each sensor from the fluorescence spectra in water, methanol, and so on (see the Supporting Information). 3) The position of a sensor near micellar membranes is altered by changing its substituents R–R. The sensor bearing more hydrophilic substituents is expected to stay at a more hydrophilic region in the nanospace. Finally, by collecting the environmental data for 1– 18, proton concentration maps near micellar membranes can be established in the form of DpKa–e diagrams. In the present study, Triton X-100 (neutral, radius: < 4.8 nm), octyl b-dglucopyranoside (OG; neutral, ~ 2.3 nm), sodium dodecylsulfate (SDS; anionic, < 3.6 nm), and cetyltrimethylammonium chloride (CTAC; cationic, < 3.5 nm) are used as micelle media in which the nanoscaled proton gradients are evaluated. The fluorescence properties of 9 in water and 18 in Triton X-100 aqueous solution during titrations are shown in Figure 1 as representatives of sensory functions. Regarding proton concentration, the DpKa value for 18 in the Triton XScheme 1. Fluorescent multiplexing sensors 1–18. The orders of 1!9 and 10!18 are determined by the logP (n-octanol/water partition coefficient) value of a corresponding amine RRNH (see the Supporting Information).

Quantitative mapping of aqueous microfluidic temperature with sub-degree resolution using fluorescence lifetime imaging microscopy

The use of a water-soluble, thermo-responsive polymer as a highly sensitive fluorescence-lifetime probe of microfluidic temperature is demonstrated. The fluorescence lifetime of poly(N-isopropylacrylamide) labelled with a benzofurazan fluorophore is shown to have a steep dependence on temperature around the polymer phase transition and the photophysical origin of this response is established. The use of this unusual fluorescent probe in conjunction with fluorescence lifetime imaging microscopy (FLIM) enables the spatial variation of temperature in a microfluidic device to be mapped, on the micron scale, with a resolution of less than 0.1 degrees C. This represents an increase in temperature resolution of an order of magnitude over that achieved previously by FLIM of temperature-sensitive dyes.

Magnetic field effects on the fluorescence of intramolecular electron-donor-acceptor systems

Abstract The external magnetic field effects on the exciplex fluorescence of α-(4-dimethylaminophenyl)-ω-(9-phenanthryl)alkanes have been studied by photostationary, time-resolved, and magnetic field modulation fluorescence spectroscopy. The singlet-triplet degeneracy of the radical ion pair is suggested to occur at a methylene chain length of about ten.

Highly thermosensitive Ca2+ dynamics in a HeLa cell through IP3 receptors

Intracellular Ca2+ distribution and its dynamics are essential for various cellular functions. We show with single HeLa cells that a microscopic heat pulse induces Ca2+ uptake into intracellular stores during heating and Ca2+ release from them at the onset of recooling, and the overshoot of Ca2+ release occurs above the critical value of a temperature change, which decreases from 1.5 to 0.2 °Con increasing the experimental temperature from 22 to 37 °C. This highly thermosensitive Ca2+ dynamics is probably attributable to the altered balance between Ca2+ uptake by endoplasmic reticulum Ca2+‐ATPases and Ca2+ release via inositol 1,4,5‐trisphosphate receptors. These results suggest that Ca2+ signaling is extremely sensitive to temperature changes, especially around body temperature, in cells expressing inositol 1,4,5‐trisphosphate receptors.

Fluorescence label studies of thermo-responsive poly(N-isopropylacrylamide) hydrogels

Abstract Temperature-induced phase transitions and microenvironment of poly(N-isopropylacrylamide) (PNIPAM) hydrogels were studied in water using 9-(4-N,N-dimethylaminophenyl)phenanthrene (DP) as an intramolecular fluorescence probe. Fluorescence behavior of the DP-labeled PNIPAM gels depended on the conditions of the gel preparation such as concentrations of monomer and crosslinker. Thermo-responsive behavior of the PNIPAM gel was affected by copolymerization of NIPAM with a hydrophilic monomer N,N-dimethylacrylamide (DMAM) and a hydrophobic monomer methyl methacrylate (MMA). Incorporation of DMAM raised the lower critical solution temperature (LCST) of the PNIPAM gel, and that of MMA lowered it. Obtained results indicate that the NIPAM–DMAM copolymer gels with higher LCST are of a more open, water-swollen nature above their LCST and the NIPAM–MMA copolymer gels with lower LCST are of a less open, water-shrunken nature below their LCST than that of the NIPAM homopolymer gel.

SOLVENT- AND CONFORMATION-DEPENDENT ELECTRON TRANSFER INTERACTIONS IN FLEXIBLE BIAROMATIC COMPOUNDS : THE CASE OF 9-( DIMETHYLANILINO) PHENANTHRENE

Abstract Absorption- and solvent-dependent fluorescence transition moments (Ma and Mf, respectively) and energies of two differently twisted 9-(dimethylanilino)phenanthrenes are determined and compared with phenanthrene properties and with quantum chemical calculations (AM1 and CNDO/S-CI) to derive the electronic and molecular structure of the first excited singlet state (S1). The increase of Mf on increasing solvent polarity can be attributed to a solvent-induced change of the S1 nature from the phenanthrene 1 L b to 1 L a type. The observed relation Mf>Ma points to enhanced coupling of the zeroth-order electron transfer state with the ground state due to a relaxation towards planarity in accordance with the calculation. Further increase of solvent polarity leads to a strong decrease of Mf below Ma for the more twisted compound explainable with enhanced electron transfer interactions at the expense of 1 L a character associated with a narrower perpendicular rotamer distribution in S1.

Fluorescence probe studies of thermosensitive N-isopropylacrylamide copolymers in aqueous solutions

Abstract Temperature-induced phase transitions of some N−isopropylacrylamide (NIPAM) copolymers in aqueous solutions were studied with 9-(4-N,N− dimethylaminophenyl)phenanthrene (DP) as an intramolecular fluorescence probe. Lower critical solution temperature (LCST) of DP-labeled NIPAM copolymers depends on the properties and contents of comonomers incorporated in the NIPAM copolymers, i.e., a hydrophobic comonomer such as methyl methacrylate (MMA) lowers the LCST, and a hydrophilic comonomer such as methacrylic acid (MAA) raises it. The higher contents of comonomers, the bigger changes of the LCST. And the micro-environments of the NIPAM copolymers in aqueous solutions also depend on them. The temperature-induced phase transitions of NIPAM copolymers with N-n−propylacrylamide (NNPAM), and N−isopropylmethacrylamide (NIPMAM) appear to be very sharp as well as that of NIPAM homopolymer aqueous solution and their LCST values are good correlated with their copolymer compositions.

Synthesis, characterization and cellular internalization of poly(2-hydroxyethyl methacrylate) bearing α-D-mannopyranose

2-(α-D-Mannopyranosyloxy)ethyl methacrylate (ManEMA) was synthesized in stereochemically pure form, and isolated as an easy-to-handle dry white solid. Poly(ManEMA-co-HEMA)s were prepared by conventional radical copolymerization of ManEMA with 2-hydroxyethyl methacrylate (HEMA). The monomer reactivity ratio rManEMA and rHEMA were estimated to be 1.00 and 0.50, respectively. The initial clustering rate of poly(ManEMA-co-HEMA) with a mannose-binding lectin, concanavalin A, evaluated by turbidimetric assay, increased with increasing mole fraction of ManEMA units in the polymer, even at constant ManEMA unit concentration. This cluster glycoside effect of the linear glycopolymer, poly(ManEMA-co-HEMA), could be well interpreted by the sequence distribution of ManEMA units calculated on the basis of the terminal model. The cellular uptake and cytotoxicity of fluorescently labeled poly(ManEMA) was examined in HeLa cells. Confocal laser scanning microscope imaging revealed the endocytotic internalization of poly(ManEMA) in HeLa cells. The survival rate of HeLa cells treated with poly(ManEMA) was estimated to be 95.7 ± 1.7% versus untreated cells. These results indicate that ManEMA is a promising monomer for development of medicinal glycopolymers.

Conformational relaxation dynamics of a poly(N-isopropylacrylamide) aqueous solution measured using the laser temperature jump transient grating method

We observed phase transition and phase relaxation processes of a poly(N-isopropylacrylamide) (PNIPAM) aqueous solution using the heterodyne transient grating (HD-TG) method combined with the laser temperature jump technique. The sample temperature was instantaneously raised by about 1.0 K after irradiation of a pump pulse to crystal violet (CV) molecules for heating, and the phase transition was induced for the sample with an initial temperature just below the lower critical solution temperature (LCST); the following phase relaxation dynamics was observed. Turbidity relaxation was observed in both the turbidity and HD-TG responses, while another relaxation process was observed only in the HD-TG response, namely via the refractive index change. It is suggested that this response is due to formation of globule molecules or their assemblies since they would have nothing to do with turbidity change but would affect the refractive index, which is dependent on the molar volume of a chemical species. Furthermore, the grating spacing dependence of the HD-TG responses suggests that the response was caused by the counter propagating diffusion of the coil molecules as a reactant species and the globule molecules as a product species and the lifetime of the globule molecules ranged from 1.5 to 5 seconds. Thus, we conclude that the turbidity reflects the dynamics of aggregate conditions, not molecular conditions. The coil and globule sizes were estimated from the obtained diffusion coefficient. The sizes of the coil molecules did not change at the initial temperatures below the LCST but increased sharply as it approaches LCST. We propose that the coil-state molecules associate due to hydrophobic interaction when the initial temperature was higher than LCST minus 0.5 K and that the globule-state molecules generated from the coil-state molecules showed a similar trend in temperature. The phase transition was also induced by heating under a microscope, and the relaxation process was followed using the fluorescence peak shift of a fluorescent molecule-labeled PNIPAM. The result also supports the existence of a globule molecule or its assembly remains for several seconds in the phase relaxation.