Preparation Of Superparamagnetic Microbeads And Its Application On Detection Of Mite Allergy And Genetically Modified Organisms

Superparmagnetic microspheres, which are the superparamagnetic nanopatricles using nano-technology coated on the polymer material. Superparmagnetic microspheres have many characteristics:superparamagnetic, active groups and so on, which has been widely used in biotechnology and life sciences.The paper prepared superparamagnetic microspheres; it meets the requirements of bio-magnetic separation. Using the green fluorescent protein (GFP) initially verifies the characteriase of magnetic microspheres. Detailed studies on the application of magnetic microspheres in detection of mite allergen specific IgE antibodies and DNA fragment. Main findings of the study are as follows:1. Synthesis of three sizes of superparamagnetic microspheres, which were 50nm, 200nm and 1μm. They were characterized using a series of methods such as scanning electron microscope and infrared spectroscopy. The results showed that they were all the rules of the spherical shape, uniform size, and good monodispersity. The surfaces of microspheres were modificatied by carboxyl functional groups, and they had good magnetic response. These characteries meet the requirements of bio-magnetic separation technonlogy.2. Conjugation of GFP antibodies onto the prepared magnetic microspheres, capturing GFP from the crude protein, and then using the chip scanner to analysis fluorescence intensity of GFP. The results showed that superparamagnetic microspheres could couple with the GFP antibody; it also showed that with the increase of concentration of GFP, values which were enriched with superparamagnetic microspheres increased too. In addition, using the method of enrichment with immuno-superparamagnetic microspheres, GFP could be separated and purified specially.3. Studied anti-human IgE antibodes were coupled with three sizeds of superparamagnetic microspheres, made immuno-superparamagnetic microspheres. Compared three sizes of different magnetic microspheres, the results showed that they are all superior to conventional testing; among them, it is evident that the detected values of 200nm and 1μm magnetic microspheres are the best. In addition, the optimal amount of immuno-superparamagnetic microspheres to detect IgE of serum was acquired. Based on the above, invented a method to detect the specific IgE antibodes (sIgE) with immuno-superparamagnetic microspheres, it improved the sensitivity in the low concentration of IgE especially. It also showed that in the case of the total amount of the IgE was determined, when a threefold increase in the volume of the sample, the sensitivity was not been affected; the values decreased, while the volume of sample increased steadily, the value is similar to the value of the conventional method until an increased to ten times.4. Moreover, another method using immuno-superparamagnetic microspheres as a carrier to detect specific IgE antibodies was invented, directly coupled household dust mite allergen protein onto microspheres, and then detected specific IgE antibodies. The method showed to be sensitive, accurate and linear regression (R2=0.9874) in the concentration of 0.035 IU/mL-3.5 IU/mL, correlation coefficient is good.5. Detected specific DNA sequences using Ubi-1 promoter sequence of maize as object of study, the Ubi-1 promoter was commonly inserted into Genetically Modified Organism. Prepared microspheres probe, through coupling amino-modified DNA to carboxyl-modified magnetic microspheres, and then captured the specific gene sequences. The effect of enrichment with microsphere probes was tested through chip scanner, from the results, the fluorescent values were proportional to the number of copies. It has been found that longer DNA fragments coupled with microspheres, more obvious the effects of detection.