Investigations On The Separation/Purification And Detection Of β-Casein In Complex Matrices

Proteins play vital roles in life activities,and the rapid and efficient protein separation/purification is the key procedure in proteomics research.In present study,the separation/purification and detection of β-casein in complex matrices are acheved by using functional magnetic nanomaterials and rare-earth element-based fluorescent probe,which broaden the application of magnetic materials and rare-earth elements in life sciences.Titanium（Ti）and zirconium（Zr）ions are co-functionalized on the surface of Fe₃O₄@PDA nanospheres via a hydrothermal synthesis method,and the obtained Fe₃O₄@PDA-Ti⁴⁺/Zr⁴⁺ magnetic nanospheres are chacarterized with ultraviolet-visible absorption spectrum,infrared spectrum,X-ray diffraction,scanning/transmission electron microscopy and hysteresis analysis.The high affinity interaction between the Ti/Zr ions and the phosphoric acid groups in β-casein offers the Fe₃O₄@PDA-Ti⁴⁺/Zr⁴⁺ magnetic nanospheres with favorable adsorption selectivity towards β-casein.The adsorption behaviors of β-casein fits well with Langmiur model,and the adsorption capacity is 873.9 mg g^-1.The adsorbed 3-casein on the magnetic nanosphere can be effectively eluted by using Na2HPO4(0.2 mol L^-1)as stripped reagent,giving rising to a recovery of 73%.The Fe₃O₄@PDA-Ti⁴⁺/Zr⁴⁺ magnetic nanospheres are applied to the adsorptive separation ofβ-casein from milk samples,and SDS-PAGE gel electrophoresis results indicate that β-casein of high-purity can be obtained.Based on the complexation interaction of europium ion(Eu³⁺)with the β-diketone structure of tetracycline（TC）,Eu³⁺-TC fluorescent probe with emission maxmima of 620 nm is fabricated.The phosphate groups in β-casein can coordinate with Eu³⁺ in the fluorescent probe,leading to the significant fluorescence enhancement,thus a fluorescence method is developed for quantitative determination of β-casein.Factors afftecting the detection performance of β-casein are studied detailedly.Under the optimal conditions,β-casein in the concentration of 1.0×10⁷mol L^-1-1.4×10^-6 mol L^-1 can be accurately determined,and the detection limit is 2.0×10^-8 mol L^-1.This developed fluorescence method has been applied successfully to the detect β-casein contents in milk samples.