Construction Of Time-resolved Sum Frequency Spectrum System And Study Of Trans-membrane Kinetics In Biology

Interfacial sciences are of great importance in biological research,for example,interactions between substances and cells are involved in the cell membrane.Many biological and chemical reactions are also closely related to the interfaces.However,most biological characterization techniques cannot obtain the signal of the surface and interface due to the influence of the bulk signal.Fortunately,second order nonlinear optical techniques represented by sum frequency generation(SFG)spectroscopy and second harmonic generation(SHG)spectroscopy can be used to characterize the structure,orientation and degree of ordering of surface and interfacial molecules in situ due to their symmetry constraints and inherent specific surface and interface selectivity.SFG has been developed for nearly 30 years and successfully used in a variety of surface and interface sciences.In addition,due to the rapid physical and chemical reactions on the surface of molecules,traditional steady state SFG is difficult to measure these ultra-fast kinetic processes.Take the advantage of ultra-fast and ultra-high peak power characteristics of ultra-short pulse lasers,we can develop femtosecond time-resolved SFG.In this paper,we have done the following works focusing on the construction and application of femtosecond time-resolved SFG system: 1.Femtosecond time-resolved Pump-SFG system setupThe light sources used in our SFG system are 100 fs,800nm laser output by femtosecond amplifier and the wide-band infrared radiation(IR)pulse laser generated by the optical parameter amplification system.IR beam has overlapped with 800 nm narrow-band visible pulse in time and space at sample interfaces of interest.The 800 nm laser pulse has been broadened to ~1 ps through 4f system,which could improve the frequency resolution of the SFG spectrum.Based on this SFG system,another wavelength tunable pump femtosecond pulse laser is introduced to excite the interfacial molecules,and then the SFG is used to probe the evolution of vibrational states and the pathway of excited energy relaxation.We change the relative light path length between the pump light and the sum-frequency light source by the delay line to realize the time scanning.The system hardware control and data acquisition we build are based on the Lab VIEW virtual instrument.A computer can automatically change the delay time between the pump light and the SFG probe light,realizing the real-time data acquisition by the CCD spectrometer.Besides,the data acquisition and data readout mode of CCD spectrum are also extended,greatly improving the accuracy,stability and scalability of the experiment and shortening the experimental time.2.Transient spectra studies on chitin by using the femtosecond time-resolved SFGUsing the femtosecond time-resolved Pump-SFG system,we have studied the transfer of vibrational energy of chitin fibre and analyzed the arrangement of molecular chains of ?-and ?-chitin allotropes.The experimental results show that the relaxation of the vibrational energy in the molecule is not only directly transition from the excited state to the ground state,but there are very strong energy transfer processes between different vibration modes and in coupling processes of the same group.Through the transient SFG,we can observe the energy transfer pathways and diagnose the coupling between molecular groups,which may help us to assign the complex vibration peaks in fingerprint region.3.Studies on trans-membrane kinetics of cell-penetrating peptides(CPPs)We have studied the effect of Pep 1 on the flip-flop kinetics of lipid bilayer by using SFG.Then,we applied octadecyltrichlorosilane(OTS)-lipid monolayer system in SFG experiments to exam the structure and orientation changes of phospholipid molecules under the influence of Pep 1.The results showed that the flip-flop process of the lipid bilayer had become much faster under the influence of Pep 1.And we also found that the density of phospholipid molecules in the inner and outer lipid layers had appeared to be asymmetric during the peptide interaction process.This asymmetric density of phospholipid molecules may be the direct reason of triggering endocytosis and producing lysosomal vesicles.We also measured trans-membrane kinetics by using SHG,confirming that the flip-flop of lipid bilayers is directly related to the process of substance entering cell.