Protein immobilization without purification via dip-pen nanolithography

Fabrication of a nitrilotriacetic acid (NTA)/Ni²⁺ pattern by dip-pen nanolithography (DPN) for the effective immobilization of His-tagged target protein was reported. The fabrication of a nickel pattern, onto which the His-tagged protein can selectively immobilize, is the critical step for the immobilization without purification of the target protein. The immobilization of the His-tagged protein was confirmed by the selective immobilization of the His-tagged enhanced green fluorescent protein (EGFP) from Escherichia coli cell extracts without prior purification. Selective binding suggested that the NTA/Ni²⁺ patterns can be effectively used for in-situ immobilization of His-tagged proteins from protein mixtures, especially from cell lysates. This novel approach can also be applied to the creation of multiple protein patterns with various proteins by combining with the multiple spotting techniques (MIST).