Highly sensitive analytical methods are urgently needed for measuring tiny amount of analytes for analytical applications. Protein microparticles composed of alkaline phosphatase (AP) and bovine serum albumin (BSA) conjugated with neutravidin (NA) were fabricated using co-precipitation method as biolabels for analytical detection. Such microparticles preserved enzymatic activity, specific binding affinity and multiple biological functions....[ Read more ]

Highly sensitive analytical methods are urgently needed for measuring tiny amount of analytes for analytical applications. Protein microparticles composed of alkaline phosphatase (AP) and bovine serum albumin (BSA) conjugated with neutravidin (NA) were fabricated using co-precipitation method as biolabels for analytical detection. Such microparticles preserved enzymatic activity, specific binding affinity and multiple biological functions.

The enzyme characteristics and affinity of the protein microparticles were examined by colorimetric assays with p-nitrophenyl phosphate (pNPP) substrate and binding assays with biotinylated BSA. This proved the microparticles have preserved enzymatic properties and specific affinity. Zeta titration experiment proved the successful conjugation of NA on AP-BSA microparticles.

Apart from the biological functions, the physical properties and the protein compositions of the microparticles were characterized. Protein microparticles (300-1200 nm) were fabricated and characterized with particle size analyzer. Surface morphology of the microparticles was characterized with scanning electron microscope (SEM). Zeta titration experiments were designed to demonstrate the protein mixture assembly within a single microparticle and the resulted isoelectric points of microparticles were similar to the native protein molecules. The protein molecular structure was examined by FTIR spectroscopy which proved the preservation of beta-sheet and alpha-helix structure inside AP-BSA microparticles. Two interesting AP-BSA microparticles with sizes of 600 nm and 1000 nm were measured and calculated to be 1.08 × 10⁸ and 4.14 × 10⁹ AP molecules per single particle, respectively. And their equivlent enzyme densities were found to be 0.5 and 4.14 molecules/nm³, respectively.

Multifunctional protein microparticles integrated with enzymes and neutravidins, which composed without any use of chemical crosslinking reagent, were applied to sandwich immunoassay as biolabels for the detection of M IgG. Results demonstrated multifunctional NA-AP-BSA microparticles with catalytic and specific binding abilities have a potential to become a new generation of labeling system in bioanalytical applications.