The Effect of Protein Structural Conformation on Nanoparticle Molecular Imprinting of Ribonuclease A Using Miniemulsion Polymerization

Abstract

One of the major difficulties faced in the molecular imprinting of proteins is the inherently fragile and flexible nature of the protein template which makes it incompatible with most polymerization systems. Miniemulsion polymerization is a possible approach for preparing molecularly imprinted nanoparticles, and in this study, the method of initiation, the high-shear homogenization, and the surfactant used for the polymerization reaction had been considered as possible factors that can denature the template protein, ribonuclease A (RNase A). The conformation of the protein in a miniemulsion was studied using circular dichroism (CD). It was found that redox initiation was more suitable for protein imprinting and that the addition of poly(vinyl alcohol) (PVA) as a co-surfactant had proved to be effective in preserving the template protein structural integrity. On the basis of the results of the study, polymeric nanoparticles imprinted with RNase A were prepared via miniemulsion polymerization using methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDMA) as the functional and cross-linker monomers, respectively, with the conditions of the polymerization system optimized to best preserve the integrity of the protein template. In the subsequent investigation for the recognition properties of the prepared nanoparticles through batch and competitive rebinding tests, the imprinted nanoparticles prepared through the conventional (nonoptimized) miniemulsion polymerization lacked the target specificity as displayed by those prepared under the optimized conditions. This illustrated the importance of protein structural integrity in protein imprinting.

The rational development of molecularly imprinted polymer-based sensors for protein detection

“…126,134 An alternative approach is to imprint the protein at the interface in mini-emulsion polymerisation. 204,205 Other particle-based approaches include the deposition of thin shell layers over core particles, which ensure that the imprint sites (which resemble bulk imprints) remain within close proximity to the surface. 155,166 Particle-based approaches to protein imprinting have been reviewed by Tan and Tong.…”

Improving the Heat Resistance of Ribonuclease A by the Addition of Poly(N,N‐diethylaminoethyl methacrylate)‐graft‐poly(ethylene glycol) (PEAMA‐g‐PEG)

“…The CD spectrum of the native RNase A is characterized by a significant signal in the far-UV region (200-250 nm), which is indicative of the secondary structure of the protein. [37] In addition, a less significant peak is observed in the near-UV region. A signal in this region is generally attributed to a local asymmetric environment of aromatic amino acid residues, i.e., tyrosine (Tyr) [42] and a disulfide bond [43] in the case of RNase A.…”

“…[34] In the temperature range of 75 to 100 8C, RNase A undergoes irreversible inactivation. [35][36][37] In the present study, we investigated the thermal denaturation and conformation of RNase A with or without PEAMA-g-PEG. We hypothesized that a smart cationic graft copolymer with polycationic and hydrophobic chains, which interact with the enzyme surface through electrostatic and hydrophobic interactions, would regulate the enzymatic activity and increase the stability of the enzyme under extreme conditions, while the hydrophilically polynonionic chain would exert a repulsive force on the enzyme, thereby increasing the solubility and dispersive stability of the enzyme-polymer complex.…”

Protein-imprinted materials: rational design, application and challenges

“…RNase A-imprinted polymeric nanoparticles were prepared via miniemulsion polymerization of methyl methacrylate (MMA) and EGDMA as the functional and cross-linker monomers. Poly(vinyl alcohol) (PVA), sodium dodecyl sulfate (SDS), and cetyl alcohol (CA) were used together with these organic solvent-soluble crosslinkers to form a miniemulsion in the aqueous phase [52].…”