Application Of Correlation Spectroscopy Technique In Measurement Of Biomacromolecule

The technique of correlation spectroscopy is a statistical method, in which the time serial fluctuation signals are correlated to obtain information about the process that gives rise to the fluctuations, such as diffusion coefficient, concentration, association and dissociation constant. Photon scattering correlation spectroscopy and fluorescence correlation spectroscopy are two major types of correlation spectroscopy.We have developed a compact photon scattering correlation spectroscopy instrument with low power laser. Compared with current instruments, the setup can decrease disturbance of disorder radiation by using double layer refractive index sample matching vessel, and it can increase collecting and transmitting efficiency of radiation by adopting single mode fiber with gradient refractive index lens. With a low power laser, the sample has a low scathe, and the laser and the measurement system can be integrated conveniently.The setup was calibrated with different diameter latex, and the measurable range of the setup is from several nanometers to one micron.The diffusion coefficient of bovine serum albumin (BSA) was studied by photon scattering correlation spectroscopy in different ionic strength solutions. The result showed that the diffusion coefficient of protein increased with increasing protein concentration, and that when the ionic strength increased gradually, the diffusion coefficient decreased with increasing protein concentration. The concentration dependence of BSA apparent diffusion coefficients was interpreted in the context of a two-body potential of mean force that includes repulsive hard-sphere and Coulombic interactions and attractive dispersion: with increasing ionic strength, Debye screening decreased, and protein interaction changed from repulsion to attraction, and protein aggregations was onset. From the concentration dependence of BSA apparent diffusion coefficients, protein interaction parameters have been regressed. It bears a net effective charge of -9.0e and has a Hamaker constant of 2.8k_BT. This workdemonstrated that PSCS was an effective technique of studying protein interactions.The main content of FCS work is as follows: take part in the assembly of ConfoCor 2 fluorescence correlation spectrometer that is bought from Zeiss (Germany). Compared with current instruments, the setup has two detection channels that cannot only provide correlation spectroscopy but also cross-correlation spectroscopy. An argon-ion laser (458,488 and 514nm) and two helium-neon lasers (543 and 633nm) are used for the excitation of the sample. Five laser lines can provide many selections for the dyes. The sensitivity of this instrument is high and the measuring time is little. The average measuring time for a sample is 10s, and the single molecule can be detected. The operation is very easy because the fluorescence correlation spectrometer can be controlled automatically through a computer.Two dyes Rhodamine 6G and Cy5 were used to calibrate the effective excited volume of 488nm and 633nm laser lines. The excited volumes were both less than 1fl. Dilute solutions of nucleic acid were measured by the system. The result indicates FCS can detect ultrasensitively the single molecule. The realization of single molecular detection promises it use in the nucleic acid detection.A novel method for the direct detection of specific nucleic acids without amplified by the general method of PCR is firstly presented and proved by experiments. In our method, fluorescence correlation spectroscopy ( FCS ) , cleaving nucleic acid by restriction endonucleases and two nucleic acid probes hybridization techniques are combined to distinguish the specific nucleic acids. In order to test the feasibility of the method, we measured the GMOs (genetically modified organisms) as an example. The detection principle is below: firstly, two restriction endonucleases FOK I and BsrD I are used to cleave the genomic DNA and the 169bp fragments of CaMV 35S promoter are retrieved, secondly, two nucleic acid probes labeled by Rhodamine Green and Cy5...