Immobilization of trypsin in polyaniline-coated nano-Fe3O4/carbon nanotube composite for protein digestion

In this report, a four-component nanocomposite, trypsin-immobilized polyaniline-coated Fe₃O₄/carbon nanotube composite, was synthesized for highly efficient protein digestion. Fe₃O₄ was deposited by the chemical coprecipitation of Fe²⁺ and Fe³⁺ in an alkaline solution containing carbon nanotubes (CNTs) to prepare nano-Fe₃O₄/CNT composite. Subsequently, polyaniline (PA) was assembled on the Fe₃O₄/CNT composite by the in situ polymerization of aniline in the presence of trypsin to obtain trypsin-immobilized PA/Fe₃O₄/CNT nanocomposite. The novel 1D superparamagnetic biomaterial has been characterized by TEM, SEM, XRD, and magnetometric analysis. The feasibility and performance of the unique magnetic biomaterial have been demonstrated by the tryptic digestion of bovine serum albumin, myoglobin, and lysozyme within 5 min. The digests were identified by MALDI-TOF MS with sequence coverages that were comparable to those obtained from the conventional in-solution tryptic digestion. The present biocomposite offers considerable promise for protein analysis due to its high magnetic responsivity and excellent dispersibility. It can be easily isolated from the digests with the aid of an external magnetic field. Because the enzyme-immobilized nanocomposite can be prepared by a simple two-step deposition approach at low cost, it may find a wide range of biological applications including proteome research.