Halfsphere Derivatisation of Magnetic Micro Particles

Abstract: Abstract   This exam project is an effort to derivatize one side of magnetic beads with one kind of molecule  , and another one on the opposite side. First the surface of the sphere is loaded with a suitable linker with, e.g. amino or hydroxyl groups. In the second step, these groups are derivatized with a photosensitive protecting group such as Nitroveratryloxycarbonyl. In the third step, the particles are placed on a surface and then irradiated with UltraViolet light (320 nm) from above, which will cleave off the Nitroveratryloxycarbonyl on the upper half, while leaving in place the ones at the lower half. The linker groups of the upper half can now be derivatized by other reagents of choice. The remaining Nitroveratryloxycarbonyl groups can be removed by suspending the particles in a solvent and then exposing them to UltraViolet light. Finally the linker groups on this half of the particles can be derivatized by a second reagent. Magnetic particles were marked with FITC, two different kinds of magnetic particles were selected, sikastar-NH2 function and sikastar-COOH function. Five different solvents were used to wash the magnetic particles and remove the bounded FITC, solvents are Acetone, 1-butanol, DMSO, 4-propanol, and Urea. Magnetic particles sikastar-NH2 and sikastar-COOH were washed with Tween 20 and SDS to remove non-specific binding of FITC. Sikastar particles were treated with IgG'FITC in constant presence of the following solvents: PBS'10, Pluronic-F127, Tween 20. Pegylation of sikastar particles got done to reduce non-specific binding. Derivatisation of Nitroveratryloxycarbonyl got done and specific bindning of IgG'FITC to micromer particles got done by protein thiolation. When a different concentration of FITC was tested to control specific and non-specific binding to sikastar functions, we observed that we had a specific binding to sikastar-NH2 in the lowest concentration. In choice of magnetic particles we had specific binding with sikastar-NH2. Using a different solvents Acetone, 1-butanol, 4-propanol, and Urea  to remove bounded FITC, sikastar-NH2 showed stronger fluoresence than sikastar-COOH after washing because of specific binding and it was difficult to remove FITC with Acetone, 1-butanol,  4-propanol,and Urea, on the other hand DMSO could remove bounded FITC from sikastar particles. When we washed magnetic particles sikastar-NH2 and sikastar-COOH with Tween 20 and SDS to remove non-specific binding of FITC, we could see that magnetic particles showed fluoresence in both functions due to non-specific binding. When sikastar particles got treated with IgG'FITC in constant presence of solvents PBS'10, Pluronic-F127, and Tween 20, we had a specific binding between sikastar particles and IgG'FITC in a presence of pluronic-F127. Pegylation of sikastar particles with a different kind of a PEG was possibl to reduce non-specific bindning. The derivatisation of Nitroveratryloxycarbonyl could be done in a N2 environment, and Nitroveratryloxycarbonyl-sikastar-NH2 could be radiated with UltraViolet light to remove Nitroveratryloxycarbonyl. Also thiolation method could be used to perform specific binding of IgG'FITC to micromer particles.