Protein immobilization is imperative for bioengineering and biochemical applications. Although many excellent approaches have been proposed, most require elaborate skills and labor-intensive techniques. In this work, we report that phenothiazine compounds such as thionine chloride (TC) are potential molecular adhesives for protein immobilization. According to conductive-mode atomic force microscopic analysis (C-AFM), TC exists with a specific affinity for proteins and can be modified on solid surfaces like ITO glass squares (denoted ITO|TC) via a simple diazotization-reduction process. Because the affinity is ∼200 nN, equivalent to 102 C-C single bonds, a variety of proteins including glucose oxidase (GOx), glucose dehydrogenase (GDH), ferritin (FT), bovine serum albumin (BSA), and salmon testes DNA (DNA) can thus be immobilized on ITO through TC. Most proteins, however, behave like insulators in aqueous solutions (0.1 M KCl). For this reason, the protein-treated ITO|TC electrodes exhibited insignificant electric responses to the applied bias voltage (Vs) until |Vs| > 1 V, contrasting sharply with the current-voltage curves found with bare ITO and ITO|TC. According to the Simmons model for metal|insulator|metal junctions, we estimated the energy barrier (φB) to the transport of electrons in proteins to be 1.0-1.5 eV, and the effective mass of the electrons (m*), to be 1/1000 the mass of free electrons. We also noticed that FT showed similar results on φB and m* as adsorbed on ITO|TC and ITO. TC is thus suggested not to interfere with the charge transport in the attracted proteins. Some other phenothiazine compounds were also characterized for their potential as molecular adhesives. Although their performances were not as promising as that of TC, phenothiazine compounds prove to be useful molecular glues for protein immobilization.
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