Controlled Dispersion and Purification of Protein–Carbon Nanotube Conjugates Using Guanidine Hydrochloride

Abstract

For the development of biofunctional carbon nanotubes for biosensors, drug carriers, and nanobiocatalysts, their aggregation and biofouling in aqueous solutions are crucial problems because this behavior leads to a reduction of their excellent optical and electrical properties and nanoscale size effects. This paper presents a new method for enhancing the dispersibility of protein-carbon nanotube conjugates and for exfoliating the protein from the carbon nanotube sidewalls through controlling the concentration of guanidine hydrochloride (Gdn·HCl) in the solution. In medium concentrations (2-3 M) of Gdn·HCl, the dispersibility of protein-carbon nanotube conjugates was found to be substantially increased without denaturation or aggregation of the proteins. At higher concentrations (>6 M) of Gdn·HCl, pristine carbon nanotubes were precipitated instantly as a result of dissociation of the protein. These phenomena indicate that Gdn·HCl functions not only as a dispersion adjuvant for biofunctional protein-carbon nanotube conjugates, but also as a cleaning agent for the purification of biofouled carbon nanotubes. The dissociation concentrations of Gdn·HCl were higher than the midpoint of protein denaturation, suggesting that protein adsorption on carbon nanotubes is more stable than protein folding toward Gdn·HCl.

Arginine Side Chains as a Dispersant for Individual Single‐Wall Carbon Nanotubes

“…Thus, the positive charges of the side chains of the polypeptides on the SWCNTs generate electrostatic repulsion between SWCNTs, leading to dispersion. In fact, the addition of 100 m M NaCl caused the SWCNTs to aggregate (data not shown), which is accounted for by the electrostatic screening effect 23…”

“…The dispersibility of SWCNTs using peptides and proteins also depends on pH and salt concentration 4. 19, 20, 23, 36 The dispersibility is reduced considerably at the isoelectric point or in the presence of salts. The solution used in this study was at neutral pH, without any other salts.…”

“…In fact, basic proteins, that is, arginine‐rich proteins, such as lysozyme and histones, were experimentally demonstrated to debundle or highly disperse SWCNTs 19. 23, 50 This insight into the behavior of arginine on the sidewalls of SWCNTs will be useful for the application of SWCNTs in medicine, nanotechnology, and nanotoxicology.…”

“…In fact, the addition of 100 mm NaCl caused the SWCNTs to aggregate (data not shown), which is accounted for by the electrostatic screening effect. [23] Fluorescence spectra of SWCNTs Debundled semiconducting SWCNTs are known to exhibit fluorescence in the near-infrared region. [37,38] To demonstrate the debundling of SWCNTs in our polypeptide tests, we recorded fluorescence spectra for the SWCNTs treated with PLA (> 70 k) and PLL (> 30 k) at 1.0 mg mL À1 .…”

“…20 Proteins can retain their intrinsic conformations even when bound to the sidewalls of SWCNTs 21. 22 However, such proteins can be readily affected by solution conditions, such as the presence of solutes that reduce the binding forces and dispersibility 23. The resulting bundling and flocculation of SWCNTs deteriorates the optical and electrical properties of the SWCNTs24 such that high colloidal stability, that is, high dispersibility, is required for their use in applications.…”