Some New Applications Of Resonance Rayleigh Scattering Method And The Constructions Of Fluorescent Sensors Based On Acridine Orange And Reduced Graphene Oxide

Resonance Rayleigh scattering (RRS) has become known for its sensitivity and simplicity as an analytical technique developed in recent years. This technique has been applied successfully to study the aggregation of chromophores on biological macromolecules and the determination of some metal ions, nonmetals and biological macromolecules etc. by means of ion association reactions with some dyes. Furthermore, it has been proved that RRS method is also a useful method for the determination of physicochemical constants such as β-cyclodextrin inclusion constant etc. RRS method provides new information concerning about molecular structure, size, shape, charge distribution, state of combination and so on. In this dissertation, RRS method was successfully utilized to determine the critical premicelle concentration, first critical micelle concentration, second critical micelle concentration, determine the isoelectric points of proteins and discriminate parallel-stranded G-quadruplex from DNAs with other topologies and structures. The experimental results of the new applications were satisfactory. Furthermore, the reduced graphene oxide was synthesized and characterized; the ways of the interaction between surfactants and reduced graphene oxide were studied. In addition, it constructed two simple, highly sensitive and selective fluorescent sensors based on acridine orange and reduced graphene oxide, which were used to the determination of cationic surfactants and hemin respectively.The main contents and some conclusions of the dissertation are as followings:1. Some New Applications of Resonance Rayleigh Scattering Method.(1) Determination of the Critical Premicelle Concentration, First Critical Micelle Concentration and Second Critical Micelle Concentration of Surfactants by Resonance Rayleigh Scattering Method without any Probe.The purpose of this work is to determine the values of critical premicelle concentration (CPMC), first critical micelle concentration (FCMC) and second critical micelle concentration (SCMC) of surfactants using a common spectrofluorophotometer by recording resonance Rayleigh scattering (RRS) signal without any probe. The plot of the RRS intensities at the maximum scattering wavelength (IRRSmax) versus surfactant concentrations (c) was constructed to obtain the IRRSmax-c curve. From the inflexions inIRRSmax-c curve, the CPMC, FCMC and SCMC values of a surfactant can be obtained sensitively. The FCMC of some anionic,cationic and nonionic surfactants such as sodium dodecyl sulfate (SDS), sodium dodecyl benzene sulfonate (SDBS), cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), Tween-20, and Tween-80were determined by RRS method and the values are in good agreement with those obtained from conductivity and surface tension measurements and literature values. The CPMC and SCMC of SDS and CTAB were also determined by RRS method respectively and the values conform to literature values too. Furthermore, RRS method can also be used to determine the FCMC of an amphiphilic macromolecule, hemoglobin, whose structure resembles a surfactant. From the experimental results, it is concluded that RRS method can be applied to the simultaneous determination of the CPMC, FCMC and SCMC values in a sensitive, accurate and no probe way.(2) Resonance Rayleigh Scattering Method for the Determination of Isoelectric Point of Protein.The relationship between RRS intensities (IRRS) of protein and pH value was studied by Resonance Rayleigh scattering (RRS) method in this article. It was found that IRRs increased with pH before isoelectric point, while it decreased with pH after isoelectric point, and the pH value corresponding to the maximal value o IRRS is the isoelectric point of the protein. The isoelectric points of five proteins have been determined by RRS method and the values are5.13for bovine serum albumin (BSA),5.13for human sreum albumin (HSA),7.00for hemoglobin (HGB),8.17for pepsin (PEP),4.33and4.75for ovalbuin (OVA) in BR buffer solution respectively, which are in accordance with the isoelectric point vales4.7-4.9for BSA,4.7-4.9for HSA,7.07for HGB,8.1for PEP,4.59and4.71for OVA. So, it established a new RRS method for the determination of isoelectric point of protein and the method has the advantages of easy operation, high sensitivity, without a probe and wide range of applications. Furthermore, the reasons for RRS enhancement were discussed too.(3) A Highly Sensitive Resonance Rayleigh Scattering Method to Discriminate Parallel-stranded G-quadruplex from DNAs with Other Topologies and Structures.The RRS intensities were significantly increased when parallel-stranded G-quadruplex was added to Mg2+solution owing to the formation of guanine nanowires (G-wires) superstructure polymer between them. But the RRS spectrum and intensity could not be changed by adding single strand, linear duplex strands, triplex strands, i-motif, and antiparallel-stranded G-quadruplex to, as Mg2+cannot change the structure of these oligomers. So, it can be used for the construction of a new RRS method to discriminate parallel-stranded G-quadruplex from DNAs with other topologies or structures. The method has high sensitivity and only1.9nM c-myc (one of the most commonly malfunctioning genes in the promoter regions of human cancers oncogenes) of parallel-stranded G-quadruplex can make a change of RRS spectrum and intensity. In addition, the RRS method can not only discriminate parallel-stranded G-quadruplex from DNAs with other topologies and structures but also recognize nanoscale parallel-stranded G-quadruplex in mixed oligomer samples. Furthermore, this RRS method was also successfully used for the study of structural transition from antiparallel-stranded to parallel-stranded G-quadruplex, and the G-wires formation of d(G4T4G4) induced by Ca2+. Therefore, by simply measuring the changes of RRS intensities, a highly sensitive RRS method that can discriminate parallel-stranded G-quadruplex from DNAs with other topologies and structures was established. 2. The Construction of Fluorescent Sensors Based on Acridine Orange and Reduced Graphene Oxide.(1) Synthesis and Characterization of Reduced Graphene Oxide and a Turn on Fluorescent Sensor based on Acridine Orange-Reduced Graphene Oxide Complex for the determination of Cationic SurfactantsThe reduced graphene oxide (rGO) were synthesized and characterized by reducing graphene oxide through hydrazine hydrate and the characteristics of rGO were conformed to the reports of literatures. Furthermore, as the special structures of surfactant and through the studies on the interactions of surfactants and the complex formed by acridine orange and rGO, it draw a conclusion that the order of interaction within surfactants and rGO was electrostatic interaction> π-π stacking interaction> hydrophobic interaction. The ways of the interaction between surfactant and rGO can guide the reactions of rGO with other substances such as oligonucleotide. In addition, it constructed a turn on fluorescent sensor based on AO-rGO platform for the determination of cationic surfactants. AO can be bound on rGO to form AO-rGO complex through electrostatic and π-π stacking interactions, resulting in the effective fluorescence quenching of AO and the "off" state of the fluorescent sensor. When the AO-rGO complex is reacted with surfactants (such as cationic surfactants CDBAC, CPB, Zeph, CTAB; nonionic surfactant TX100, TX114; anionic surfactant SDS, SDBS, SLS ect), only cationic surfactants can turn on the fluorescent sensor and the other surfactants cannot affect the fluorescence of the sensor. The AO-rGO complex will decompose because of a competitive binding of cationic surfactants with rGO, and a more stable cationic surfactants-rGO complex than AO-rGO complex is formed, resulting in the release of AO molecules from rGO surface and the "on" state of the fluorescent sensor. The fluorescent sensor can selectively react with cationic surfactants and other surfactants such as nonionic and anionic surfactants cannot affect the fluorescence of the sensor. In a certain concentration range, the recovered intensity of fluorescence is linear with the concentration of cationic surfactant. In conclusion, it constructs a relatively sensitive, selective and simple fluorescent sensor, which can be used selectively for the determination of cationic surfactants. (2) Label-free DNA Acridine Orange and Reduced Graphene Oxide-based Fluorescent Sensor for Highly Sensitive and Selective Detection of Hemin.G-quadruplex structure aptamer (PS2.M) can capture acridine orange (AO) from reduced graphene oxide (rGO) of AO-rGO complex. When the AO-PS2.M/rGO mixture is incubated with hemin, the specific binding of hemin with PS2.M results in a release of AO from PS2.M and return of AO back to rGO. Based on the quenching of fluorescence, the target hemin was detected sensitively and selectively, giving a detection limit of50nM.