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Peroxynitrite (ONOO - ), a kind of reactive oxygen species, plays an indispensable role in many physiological processes. The stability and reactivity of ONOO - are significantly affected by the pH of the environment. A novel fluorescent probe RN-NA that can simultaneously respond to ONOO - and pH was proposed and constructed based on a rational-designed multifunctional fluorescence resonance energy transfer (FRET) platform. The RN-NA probe exhibited a remarkably different fluorescence change in response to ONOO - and pH. The fluorescence signals at 525 and 710 nm increased about 4-fold with a pH change from 8.0 to 3.0. The changes in fluorescence at 525 nm are mainly attributed to photo-induced electron transfer, and the fluorescence enhancement at 710 nm was mainly due to acid-induced open-closed circulation. In the presence of ONOO - , the fluorescence at 525 nm increased 5-fold, while the fluorescence at 710 nm was almost completely diminished. Up to 70-fold fluorescence enhancement was observed in the ratiometric channel F 525 / F 710 . In the cell imaging experiment, the intracellular pH was adjusted using H + /K + ionophore and nigericin, and the endogenous ONOO - was generated by lipopolysaccharide (LPS) and γ-interferon (IFN-γ). The RN-NA probe can respond to cellular pH and endogenous ONOO - with remarkable fluorescence changes in both red/green and ratiometric channels.

Two molecules, 7-(diethylamino)coumarin-3-carbohydrazide (DCCH) and fluorescein-5-thiosemicarbazide (FTSC) were investigated in different solvents, under varying pH conditions regarding their spectroscopic properties for the usage as a Förster Resonance Energy Transfer (FRET) pair to study the molecular interaction between cellulosic surfaces. All the relevant spectroscopic properties to determine the Förster distance were measured and the performance as a FRET system was checked. From the results, it is clear that the environmental conditions need to be accurately controlled as both, but especially the FTSC dyes are sensitive to changes. For high enough concentrations positive FRET systems were observed in DMF, DMSO, H2O, THF and alkaline DMF. However due to the low quantum yield of the unmodified DCCH throughout the investigated parameter range and the strong environmental dependency of FTSC, both dyes are not preferable for being used in a FRET system for studying interaction between cellulosic surfaces., Competing Interests: The authors have declared that no competing interests exist.

The determination of cellulase distribution on the surface of cellulose fiber is an important parameter to understand when determining the interaction between cellulase and cellulose and/or the cooperation of different types of cellulases during the enzymatic hydrolysis of cellulose. In this communication, a strategy is presented to quantitatively determine the cellulase colocalization using the fluorescence resonance energy transfer (FRET) methodology, which is based on acceptor photobleaching and spectrally unmixing fluorescence microscopy. FRET monitoring of cellulase colocalization was achieved by labeling cellulases with an appropriate pair of FRET dyes and by adopting an appropriate FRET model. We describe here that the adapted acceptor photobleaching FRET method can be successfully used to quantify cellulase colocalization regarding their binding to a cellulose fiber at a resolution <10 nm. This developed quantitative FRET method is promising for further studying the interactions between cellulase and cellulose and between different types of cellulases.

A novel real-time in situ detection method for the investigation of cellulase-cellulose interactions based on fluorescence resonance energy transfer (FRET) has been developed. FRET has been widely used in biological and biophysical fields for studies related to proteins, nucleic acids, and small biological molecules. Here, we report the efficient labeling of carboxymethyl cellulose (CMC) with donor dye 5-(aminomethyl)fluorescein and its use as a donor in a FRET assay together with an Alexa Fluor 594 (AF594, acceptor)-cellulase conjugate as acceptor. This methodology was successfully employed to investigate the temperature dependency of cellulase binding to cellulose at a molecular level by monitoring the fluorescence emission change of donor (or acceptor) in a homogeneous liquid environment. It also provides a sound base for ongoing cellulase-cellulose study using cellulosic fiber.