| With the completion of Human Genome Project ahead of the time, biologic analysis technique has entered a gene times. The detection method of biomacromolecule has developed rapidly which has become the forward field of life science. The common analytical methods mainly include spectrophotometry and fluorimetry, but they have some serious disadvantages. Spectrophotometry suffers from low sensitivity, multiple steps, and long time; fluorimetry is restricted by fluorimetric systems and fluorimetric reagents are toxic and expensive. So far there isn't any perfect analytical method to detect nucleic acid and proteins. Resonance light scattering (RLS) technique is a new analytical method with high sensitivity, simplicity, rapidity, and good selectivity.The research of the interaction between the small molecule and the nucleic acids has aroused general concern recently. So the work we have done not only establishes the effective analytical method but also helps us to discover the mechanism of pathological change and develop new anti-cancer medicine, thus it has great significance. Small molecule combines with nucleic acid by three modes: intercalative binding, groove binding and electrostatic force.In this dissertation eight new RLS methods were established and the relative mechanism was discussed.Firstly, tetraphenyl porphyrin cobalt chlorine (CoTPPCl) was utilized to determinate nucleic acid. Nucleic acid enhances the weak RLS signal of CoTPPCl under the optimum conditions, and the enhanced light scattering intensity is proportional to the concentration of nucleic acid in the range of 0.05—3.5μg/mL for calf thymus DNA, 0.03—4.2 μg/mL for fish sperm DNA and 0.02—4.9μg/mL for yeast RNA. The detection limits are 3.5 ng/mL for calf thymus DNA, 4.5 ng/mL for fish sperm DNA and 6.1 ng/mL for yeast RNA, respectively. Synthetic samples were determined with satisfactory results.Secondly, a new RLS method for the determination of nucleic acid was developed by using tetraphenyl porphyrinatoiron chlorine (FeTPPCl). FeTPPCl binds with nucleic acid by intercalation and aggregates on the surface of nucleic acid to enhance RLS signals. The proposed method is more sensitive than that of CoTPPCl.Thirdly, u-oxotetraphenyl porphyrinatoiron [(FeTPP)2O] was employed to determinate nucleic acid. Besides coordinating with nitrogen atoms on the porphyrin cycle, the iron ion combines with the oxygen atom, thus the radii of the iron ion reduces so it can coordinate with the porphyrin cycle in the same plane. The rigidity and coplanarity of the molecule are improved greatly, which is profitable to the RLS enhancement. The linear ranges are 0.02—2.5 ug/mL for calf thymus DNA, 0.02—2.7 ug/mL for fish sperm DNA, and 0.03—3.1 u.g/mL for yeast RNA. The detection limits (3a) are 1.0 ng/mL for calf thymus DNA, 1.1 ng/mL for fish sperm DNA and 1.2 ng/mL for yeast RNA, respectively. In comparison with mononuclear metalloporphyrin, binuclear metalloporphyrin is more sensitive.Fourthly, a new method has been developed for determination of DNA by resonance light scattering with zwitterionics Dodebyl dimethyl betaine (BS-12) in aqueous solution as a new probe. At pH9.3, the interactions of BS-12 with DNA gave strong RLS signals at 388.0nm. Linear relationships were found between the enhanced intensity of RLS and the concentration of DNAs in the range 0.15" 5.4 y g/mL for fsDNA and 0.20" 6.8 n g/mL for ctDNA. The limits of detection were 1.5and 1.6ng/mL for fsDNA and ctDNA, respectively.Fifthly, zwitterionics cocamidopropyl hydroxysultaine (HSB) was firstly used to determinate DNA. HSB has one cationic group and several anionic groups. After nucleic acid is added, the nitrogen atom of HSB combines with the negative phosphate groups of nucleic acid. Apart from this, the anionic group of zwitterionics binds with the positive bases of nucleic acid. The linear range is 0.02—4.25 ug/mL and the detection limit (3a) is 1.5 ng/mL for calf thymus DNA. Compared with those of cationic surfactants, the proposed method has a wider linear range and lower detection limit.Sixthly, a novel assay of proteins was established based on enhanced RLS signal of ammonium molybdate. Under optimum conditions ammonium molybdate reacts with proteins to form a new supermolecule complex. The molecule weight enhancement of scattering particles and the hydrophobicity of supermolecule complex enhance RLS signal. The enhanced RLS intensity is proportional to the concentration of proteins in the range of 0.25—8.5 ug/mL for bovine serum albumin and 0.40—10.2 ng/mL for human serum albumin. The detection limits (3a) are 12.5 ng/mL for bovine serum albumin and 14.4 ng/mL for human serum albumin. The results of the determination for human urine and serum samples are close to those obtained from theCoomassie Brilliant Blue method.Seventhly, phosphotungstic acid was employed to determinate proteins in the presence of dodecylbenzenesulfonate sodium by the RLS technique. Phosphotungstic acid combines with protein, and then dodecylbenzenesulfonate sodium forms a micelle on the surface of phosphotungstic acid-protein. It reduces the distance of phosphotungstic acid and protein, and enlarges the volume of scattering particles. The formation of micelle is helpful for the RLS enhancement. The linear ranges of bovine serum albumin and human serum albumin are 0.07— 4.5 |4.g/mL and 0.08—4.9 ng/mL. The detection limits (3a) are 6.8 ng/mL for bovine serum albumin and 7.9 ng/mLfor human serum albumin. The proposed method is more sensitive than that of ammonium molybdate.Eighthly, a new method for the determination of DNA was developed with Azocarmine G (AG) in the presence of cetyltrimethylammonium bromide (CTMAB) by the resonance light scattering (RLS) technique. In the buffer solution(pH9.0), the positive charge CTMAB will continue to assemble on DNA to form positive charged pre-micella, which promotes the assembly of negative charged AG on the pre-micella and form three components of DNA-CTMAB-AG associate. During the formation of associate, the surfactant CTMAB acts as the mediator for which draws close the distance between DNA and AG, which enhance their interaction and cause enhanced RLS. The enhanced RLS intensity was proportional to the concentrations of DNAs in the range 0.03" 1.5 u g/mL for fsDNA and 0.02" 1.5 y g/mL for ctDNA. The detection limits were 2.8, 2.0 ng/mL for fsDNA and ctDNA, respectively. Compared with other methods, this method has large detective wavelength, which can deduce the interference of background and increase the intensity of resonance light scattering.The chief characteristics of this dissertation are as follows:1. We studied three metalloporphyrin-nucleic acid systems, analyzed their difference and established three new assays of nucleic acid. We firstly used metalloporphyrins to determinate nucleic acid and found that there exists a rule for their sensitivities: binuclear metalloporphyrin >mononuclear metalloporphyrin >nonmetal porphyrin. It can be concluded that coplanarity of RLS probes is a key factor of sensitivities. Three metalloporphyrins bind with nucleic acid in the modes of intercalation, which offers a convictive proof of filtrating anti-cancer medicine. Thepart of the work has been published in Microchimica Acta and Canadian Journal of Analytical Sciences and Spectroscopy. The others will be published in Analytical Letters and Instrumentation Science & Technology.2. Zwitterionics was firstly performed to determinate nucleic acid. In our opinions, anionic and cationic groups of zwitterionics bind with phosphate groups and bases of nucleic acid to produce a large-sized complex by electrostatic force, which enhances RLS signals. The proposed method is more sensitive than cationic surfactants. The work has been published in Analytica Chimica Acta and Chinese Journal of Analytical Chemistry.3. We investigated two poly acid-protein systems, discussed mechanism of RLS enhancement, and proposed two new methods for the determination of protein. The common protein probes are primarily anionic dyes, but ammonium molybdate and phosphotungstic acid enlarged the range of RLS probes. For the phosphotungstic acid method, the utilization of dodecylbenzenesulfonate sodium forms a micelle and improves the sensitivity of the method. The work on ammonium molybdate has been published in Canadian Journal of Analytical Sciences and Spectroscopy and the work on phosphotungstic acid will be published in Spectroscopy Letters.4. Azocarmine G (AG) is a common acidic anion dye with n - n stacking electric properties. Azobenzene dye is a kind of sensitive RLS probe of protein, however, it has not yet been performed for the RLS determination of nucleic acid. In this work, we found that AG and DNA in the presence of cationic surfactant CTAB can form a new ternary complex AG-CTAB-DNA, which results in the RLS enhancement. Based on this, a new assay of DNA can be established. This work succeeds in extending the application range of azobenzene dye. Besides the assay of protein, it can be utilized for the determination of DNA. Compared with others, this proposed RLS method efficiently reduces background interference due to red shift of analytical wavelength, and it can gain much purer scattering light to improve its sensitivity. This method is simple, sensitive and practical. The work will be published in Journal of Central South University (English). |
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