Study On The Quantitative Detection Method Of Surface Amino Group On Silica Coated Magnetic Microspheres

The purpose of this paper is to establish a method for amino group density of silica magnetic microspheres. On the basis of literature searching fluorescamine method and ITL/BCA method is chosen.In this paper, the fluorescamine method is established to detect the surface amino group of magnetic microspheres using the amino group modified silica magnetic microspheres prepared by our lab. The detection conditions are optimized after a careful investigation of the optical properties of the reaction between fluorescamine and amino groups. We discover that the fluorescent signal is most stable when the pH of the buffer is adjusted to 8.0. Under alkaline conditions the modified aminosilane carrying amino groups will be dissociated from the surface of microsphere after 60h hydrolysis reaction, which renders a stable fluorescent signal after a further reaction of 20min with fluorescamine. And the Coefficient of variation(CV) for within-run assays is 10%,CV for between-run assays is 25%, the lowest limit of detection of this assay is 0.074mmol/g. This method is simple and fast with a high accuracy.Another method using traut's reagent and bicinchoninic acid(ITL/BCA method) to quantitatively determine the surface amino group of magnetic microspheres is also established. We choose 60℃, one hour of reaction time and 40mM of ITL concentration as the most optimum reaction condition. The difference between BCA reagent and MicroBCA reagent is studied. And the CV for within-run assays is 5%,CV for between-run assays is 10%, the lowest limited detection of this method is 0.0036mmol/g.Finally, on the basis of ITL/BCA, a new subtraction method is developed. This method successfully avoids the interference from silica magnetic microspheres. It is shown that the two methods to for evaluating the amino group density of the silica magnetic microspheres have similar results.