Fluorescent Behavior Of Lignin And The Fabrication Of Lignin-based Fluorescence Resonance Energy Transfer System

Lignin is a kind of natural fluorescent polymer materials which is also the main source of the fluorescence of plant cell walls.However,the fluorescence mechanism of lignin is still elusive until now,and there is little reference available on the functional material based on the fluorescent feature of lignin.It is of significance to study lignin fluorescent behavior and its application.In this dissertation,the complex fluorescence mechanism of lignin was revealed by studying the influence of lignin aggregation on its corresponding fluorescence,and the analytical method of lignin fluorescence was established.On these bases,the fluorescence resonance energy transfer(FRET)system with lignin as the fluorescent donor was fabiricated to promote the functional and high-valued applications of lignin fluorescence materials.Firstly,steady-state and transient fluorescence techniques were used to study the fluorescent charactereistics of lignin,including multi-fluorophore luminescence and three-dimensional fluorescence spectra.Combining the structure characteristics of lignin with the theory of aggregation through-space conjugation(ATSC),it was found that the ATSC of phenylpropane units was the origin of the long wavelength fluorescence of lignin.The loose micelles of lignin contributed to the steric hinderance of fluorophores in the aggregation and collision among lignin micelles.Consequently,the dynamic or static flurescence quenching of excited states was inhibited.The concentration-induced flurescence quenching of lignin was mainly caused by the inner-filter effect.The fluorescent intensity of lignin was enhanced by the intra-micelle aggregation of lignin,resulting from the restriction of intramolecular motion and the increasement of ATSC effect.Although the swelling lignin micelle belonged to aggregation-enhanced-emission fluorophore,the aggregate fluorescence behavior of lignin wouldgraduallytranferedfromaggregation-enhanced-emissionto aggregation-caused-quenching with the continually increasing micelle compactness,because of the decreasing steric effect of fluorophores.Based on results above,lignosulfonate(ELS)was used as the water-soluble carrier and fluorescence donor,while spirolactam Rhodamine B(SRhB)worked as the recognition element for H~+and acceptor group,and diethylenetriamine acted as the linking arm for donor and acceptor.A lignin-based FRET pH fluorescent probe(L-SRhB)was constructed by grafting the functional molecule of SRhB into the ELS macromolecule.The basic performance of L-SRhB,such as ratiometric fluorescent response,reversible response,selective response,photobleaching,and photostability were studied.It was found that the pH response range and pKa of L-SRhB were mainly determined by the property of SRhB.L-SRhB exhibited good reversible and selective response to H~+as well as a good photostability.However,the performance of photobleaching of L-SRhB needed to be further improved because of the low stability of ELS against photobleaching.In addition,the cell-imaging of L-SRhB probe was investigated by the laser scanning confocal microscopy.The results showed that ELS had no inhibitory to the cell viability within a certain concentration range,whereas the cytotoxicity of L-SRhB was significantly enhanced.L-SRhB was internalized by endocytosis pathway and could be used to distinguish normal cells from cancer cells by different cell staining rates.Considering the complexity of chemical grafting,a non-covalent lignin-based FRET system(SRhB/ELS)was constructed by self-assembly method.SRhB nanoparticles(SRhB NPs)were used as the model compound.The quantitative introduction of SRhB NPs into ELS was achieved.The particle size,morphology,bonding method,self-assembly driving force and adsorption behavior of SRhB/ELS were investigated by dynamic light scattering,scanning electron microscopy,nuclear magnetic resonance spectroscopy,Zeta potential and quartz crystal microbalance with dissipateon,respectively.It was found that SRhB NPs were adsorbed on the surface of ELS micelle by electrostatic force.The particle size of SRhB/ELS was about 100 nm.With the assistance of ELS,a water-soluble carrier,the redispersion of SRhB NPs was enhanced.The influence mechanism of ELS on the fluorescent behavior of SRhB was discussed by studing the FRET behavior of SRhB/ELS and the pH-response behavior of the complex system as a function of ELS content.The results showed that there was a stable FRET efficiency between ELS and SRhB in the composite.A lignin-based ratiometric pH probe was constructed.The FRET efficiency between the donor/acceptor groups in SRhB/ELS system increased with the decrease of ELS content in the composite,resulting in the improvement of the pH resolution and the response time of SRhB/ELS to pH.However,the fluorescent intensity of SRhB was quenched with the decrease of ELS content.The study on the fluorescence energy transfer mechanism of SRhB/ELS showed that the emission energy of ELS directly transferred to SRhB from the well-overlapped fluorophore with Rhodamine B by the non-radiative transfer mechanism,while the emission energy of non-overlapping fluorophores of ELS indirectly transferred to the acceptor fluorophore by the intra-micelle energy transfer cascades in ELS.The shape of ELS fluorescent spectra almost had no change under the energy transfer process.ELS might be used as a convenient fluorescent donor material for preparing the water-soluble FRET ratiometric probe because fluorescenct characteristic of lignin kept constant at any adsorption site in energy transfer process.