Study Of Novel Biomaterials And Characterization Methods Based On Selenium Nanoparticles

Selenium, an essential trace element, which concern many biological processes, plays a very important role in living things. Nano-Se is a kind of red element Se with biological activity, it has the advantages of "four high and one low" in toxicity and biological activity, such as high bioactivity, high safety, high immunoregulation ability, high antioxidant and low toxicity, and was first synthesized and characterized by Chinese scientists. Now, red nano-se is widely used in healthy care products, animal feed and plant regulators.Bioanalysis in cellular level provides a simple and reliable method in studying medicine and biological phenomenon. For example, flow cytometry and fluorescent markers greatly enriched biological analysis of the visual effects for the medical and pharmacological study with reliable and strong evidence. It is still very important in bioanalysis that continuously improve and establish new biological detection methods according to specific requirements. So, we started electrochemical analysis research based isolated cell culture system, then developed a novel biosensor and used it into cell biological analysis. This provides a new choice for the rapid and accurate screening and detection of drug activity.This dissertation has focused on the study of preparation of red nano-se and construction of composite nano-drug agents with selenium and anticancer drug. While studying the biological activity of selenium nanoparticles, two analysis methods of detecting biological activity in the cells level was proposed and developed. The work mainly includes the following parts:1,Preparation and bioanalysis of Se nanoparticles.We improved the preparation method of Se nanoparticle by chitooligosaccharide replaced chitosan as the template to prepare the red se nanoparticles (SeNP) successfully using ascorbic acid as the reducing agent. It left out the resolve step of chitosan by acetic acid, and the toxicity of vestigital acetic acid. Then the nanoparticle was characterized by transmission electron microscopy, infrared spectroscopy, X-ray diffraction, and other means. It was showed that the nanoparticle was uniformity, well dispersion, the average size was 32 nm, and the selenium was physically adsorption in the chitooligosaccharides surface at amorphous state. When the SeNP was cultured with liver cancer cells, the result showed that the bioeffect of SeNP related with concentrations, as the low concentration promoted the cell proliferation while high concentration inhibit its growth.2,Combined method of fluorescence spectrum and thiazole blue for detecting antioxidant activity of SeNP.Based on the mechanism of hydroxyl radical quenching fluorescent dye Rhodamine B, this chapter established a method for detecting hydroxyl radical scavenging ability of selenium nanoparticles, and discussed the anti-oxidation and biological activity of Se nanoparticles combined with thiazole blue method (MTT). The results showed that: a fluorescence method was established for evaluated the selenium nanoparticles's ability to scaveng the hydroxyl radical. Within a certain concentration, there was a linear relationship of SeNP between its ability to eliminate free radicals and concentration, and the ability was much stronger than the other two antioxidants. Fluorescence-MTT assay showed biological activity of SeNP was in good agreement with Weinberg principle.3,Development and application of fluorouracil-chitooligosaccharide/se nanoparticles.Fluorouracil-chitooligosaccharide/se nanoparticles (Fu-COS/SeNP) were first prepared by two steps: 5-fluorouracil was first attached to chitooligosaccharides via acetyl, and then Se nanoparticles were absorbt on the netted texture of chitooligosaccharides. The average size of Fu-COS/SeNP was 433nm, and the nanoparticles could release 5-fluorouracil and selenium slowly. When Fu-COS/SeNP was cultured with cancer cells, we found that the activity substate of 5-fluorouracil and selenium synergistically inhibit the cell proliferation observably, this illustrated clearly that the novel complex biomaterial enlarge the antitumor effect of 5-Fu.4,Bioanalysis of Se nanoparticles by electrochemical impendance method.Based on specific recognition between Annexin V protein and phosphatidylserine which exposed on the membrane of early apoptotic cells, and Annexin V protein was immobilized on the electrode surface using the self-assembly technology, we successfully constructed a biosensor that it would be used to detect early apoptotic cells. Then the biosensor was used to detect the self-established early apoptotic cell model, the results showed that Annexin V modified biosensors could specific recognize the apoptotic cells, the electrochemical impendent signal changed obviously after recognition, and the biosensor could detect the apoptotic cells linearity after incubated with the cells 1h in HEPES buffer with 5mM Ca2+ at 37℃. The detecting results were consistent with that detected by flow cytometry and fluorescent microscope. Then the biosensor was successfully used to detect the apoptotic cells induced by SeNP and 5-Fu, this indicated that the Annexin V modified biosensor could be used for detecting the antitumor activity of biomaterials such as SeNP. Here, we provided a simple and convenientt analysis method for biomaterials screening.5,Application of chitooligosaccharide-Se nanoparticle for plant nutrition regulator.We prepared chitooligosaccharides-Se nanoparticles with improvement of preparation of selenium chitosan, and studied the formation and stability of the nanoparticles by transmission electron microscopy, zeta-sizer, et al. We found that under acid condition, selenite radical combined with active hydroxide radical and amino-group of chitooligosaccharides to form chitooligosaccharides - Se compound molecule, the remaining group complex interacted with the negative sodium tripolyphosphate via electrostatical interactation to form nanoparticles with the size about 100nm. The nanoparticle was stable below 4℃, but above 25℃, chitooligosaccharides would swell, and nanoparticles is decomposed. When the nanoparticles were used as a kind of plant nutrition regulator for rice cultivation, the results showed that the production of rice was increased, the anti-oxidation substance in rice was significantly enhanced, and the content of selenium can be reached to 0.25 mg / kg around. So the nutritional quality of selenium-enriched rice we get is much better than that of ordinary rice.