Abstract
Continued advancement of protein array, bioelectrode, and biosensor technologies is necessary to develop methods for higher amount and highly oriented immobilization activity of proteins. In pursuit of these goals, we developed a new immobilization method by combining electrostatic transport and subsequent molecular diffusion of protein molecules. Our developed immobilization method is based on a model that transports proteins toward the substrate surface due to steep concentration gradient generated by low-frequency AC electric field. The immobilization of the maximum amounts can be obtained by the application of the AC voltage of 80 Vpp, 20 Hz both for His-tagged Green Fluorescent Protein (GFP) and Discosoma sp. Red Fluorescent Protein (DsRed), used as model proteins. The amounts of the immobilized His-tagged GFP and DsRed were approximately seven-fold higher than that in the absence of the application of low-frequency AC electric field. Furthermore, the positively and negatively charged His-tagged GFP at acidic and alkaline pH were immobilized by applying of low-frequency AC electric field, whereas the non-charged His-tagged GFP at the pH corresponding to its isoelectric point (pI) was not immobilized. Therefore, unless the pH is equal to pI, the immobilization of electrically charged proteins was strongly enhanced through electrostatic transport and subsequent molecular diffusion.
Highlights
The immobilization of proteins on the surface of solid materials is a key technology in the production of protein arrays, biosensors, and bioelectrodes for use in the analytical and bioelectronics fields
His-tagged Green Fluorescent Protein (GFP) and Discosoma sp. Red Fluorescent Protein (DsRed) were immobilized on the substrate by the method combining electrostatic transport and subsequent molecular diffusion
We developed a new immobilization method by combining electrostatic transport and subsequent molecular diffusion enhanced by steep concentration gradient of protein molecules
Summary
The immobilization of proteins on the surface of solid materials is a key technology in the production of protein arrays, biosensors, and bioelectrodes for use in the analytical and bioelectronics fields. Proteins may be immobilized by physical absorption (e.g., dot blot [7,8] and polyacrylamide gel [9,10] methods), through covalent coupling or cross-linking [3,11,12,13,14,15], as a self-assembled monolayer (SAM) [16,17,18,19], or through affinity interactions (e.g., avidin/biotin [3,20,21], nickel-nitrilotriacetic acid (Ni-NTA)/His-tagged protein [3,22,23,24], and intein methods [1,25,26] These protein physical absorption/immobilization techniques were designed for immobilizing large amounts of highly active proteins at a high density.
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