Kaimin Chen

Electrostatic selectivity in protein-nanoparticle interactions.

The binding of bovine serum albumin (BSA) and β-lactoglobulin (BLG) to TTMA (a cationic gold nanoparticle coupled to 3,6,9,12-tetraoxatricosan-1-aminium, 23-mercapto-N,N,N-trimethyl) was studied by high-resolution turbidimetry (to observe a critical pH for binding), dynamic light scattering (to monitor particle growth), and isothermal titration calorimetry (to measure binding energetics), all as a function of pH and ionic strength. Distinctively higher affinities observed for BLG versus BSA, despite the lower pI of the latter, were explained in terms of their different charge anisotropies, namely, the negative charge patch of BLG. To confirm this effect, we studied two isoforms of BLG that differ in only two amino acids. Significantly stronger binding to BLGA could be attributed to the presence of the additional aspartates in the negative charge domain for the BLG dimer, best portrayed in DelPhi. This selectivity decreases at low ionic strength, at which both isoforms bind well below pI. Selectivity increases with ionic strength for BLG versus BSA, which binds above pI. This result points to the diminished role of long-range repulsions for binding above pI. Dynamic light scattering reveals a tendency for higher-order aggregation for TTMA-BSA at pH above the pI of BSA, due to its ability to bridge nanoparticles. In contrast, soluble BLG-TTMA complexes were stable over a range of pH because the charge anisotropy of this protein at makes it unable to bridge nanoparticles. Finally, isothermal titration calorimetry shows endoenthalpic binding for all proteins: the higher affinity of TTMA for BLGA versus BLGB comes from a difference in the dominant entropy term.

Binding between proteins and cationic spherical polyelectrolyte brushes: effect of pH, ionic strength, and stoichiometry.

Cationic spherical polyelectrolyte brushes (SPBs) were synthesized by photoemulsion polymerization, consisting of a polystyrene core with a diameter around 80 nm and a poly(2-aminoethylmethacrylate hydrochloride) (PAEMH) shell with a thickness from 10 to 50 nm densely grafted on the core surface. The binding of various proteins onto SPBs was observed by turbidimetric titration, dynamic light scattering (DLS), zeta potential, and isothermal titration calorimetry (ITC). The binding, aggregation, and releasing of proteins by SPB can be tuned by modulating pH. The pH regions of binding for bovine serum albumin (BSA), β-lactoglobulin (BLG), and papain onto SPBs are markedly different and tunable by ionic strength and stoichiometry between protein and SPB. Binding energetics, affinity, and amount of various proteins onto cationic SPBs were determined by ITC. These findings lay the foundation for SPB applications in the protein purification and selective immobilization of different proteins, enzymes, and antibodies.

Protein immobilization and separation using anionic/cationic spherical polyelectrolyte brushes based on charge anisotropy

Interactions between bovine serum albumin (BSA)/β-lactoglobulin (BLG) and spherical polyelectrolyte brushes (SPBs) were investigated by a combination of turbidimetric titration, dynamic light scattering (DLS), zeta potential measurement, and small angle X-ray scattering (SAXS) which revealed different behaviors, architectures, and phase states of pH dependent protein–SPB interactions. Binding energetics, affinity, and stoichiometry between BSA–BLG and SPBs were determined by isothermal titration calorimetry (ITC) to get further information concerning the interaction difference. The SPBs consist of narrowly distributed polystyrene core particles (ca. 80 nm in diameter) onto which linear chains of polyelectrolytes, either weak anionic poly(acrylic acid) (PAA) or weak cationic poly(2-aminoethyl methacrylate hydrochloride) (PAEMH), are grafted. For a particular protein (BSA or BLG), the binding stoichiometry, affinity, architecture, and phase state between proteins and anionic SPBs were significantly different from those for cationic SPBs. Significantly larger binding affinity and adsorbed amount were observed for BSA in anionic SPBs versus cationic SPBs, while opposite for BLG, which were explained in terms of different charge anisotropy of proteins. These findings lay the foundation for SPB applications in the separation and immobilization of different proteins.

Recyclable Spherical Polyelectrolyte Brushes Containing Magnetic Nanoparticles in Core

Spherical polyelectrolyte brushes consisting of a magnetite/polystyrene nanocomposite core and a poly(acrylic acid) brush shell were prepared by photo-emulsion polymerization. They are narrowly dispersed, superparamagnetic and redispersible after aggregating by external magnetic field, as determined by transmission electron microscopy, dynamic light scattering, thermal gravimetric analysis and a vibrating sample magnetometer. Magnetic control is thus introduced into nano-sized spherical polyelectrolyte brushes to achieve recovery and controllable delivery in applications. This approach opens up the way for cost-effective applications of spherical polyelectrolyte brushes.

Tunable adsorption of bovine serum albumin by annealed cationic spherical polyelectrolyte brushes.

By combining turbidimetric titration, dynamic light scattering (DLS), and zeta potential methods, we demonstrated that the adsorption of bovine serum albumin (BSA) in annealed cationic spherical polyelectrolyte brushes (SPB) can be controlled by modulating the pH, ionic strength, BSA concentration of the mixed solution, and SPB thickness. The SPB consist of a polystyrene core with a diameter around 80 nm and a dense shell of poly (2-aminoethylmethacrylate hydrochloride) (PAEMH) with a thickness from 10 to 60 nm covalently attached on the core surface. Results revealed the existence of three pH regions, corresponding to (1) adsorption of BSA in SPB, (2) aggregation of SPB induced by BSA adsorption, and (3) desorption of BSA from SPB. The extent of the pH regions can be modulated by ionic strength, BSA concentration, or SPB thickness. Adsorption measurements demonstrated that the adsorbed amount of BSA in SPB was affected by pH, ionic strength, and SPB thickness. These findings lay the foundation for protein separation by SPB.

Enhancement of enzymatic activity by magnetic spherical polyelectrolyte brushes: a potential recycling strategy for enzymes.

Interactions between amyloglucosidase and magnetic spherical polyelectrolyte brushes (MSPB) were studied by turbidimetric titration, which reveals reversible and tunable behaviors of pH-dependent enzyme-SPB binding. Quantitative thermodyanmic parameters including binding affinity and stoichiometry between enzyme and SPBs were further measured by isothermal titration calorimetry (ITC). A large amount of enzyme can be loaded in MSPB without loss of MSPB stability. We demonstrated that the enzymatic activity of amyloglucosidase bound in MSPB could be greatly enhanced (catalytic reaction rate, k(bound) = 1.36k(free)) compared to free enzyme acitivity in solution. This is tremendous improvement from other carrier systems that usually lead to a significant decrease of enzymatic activity. Both the high enzyme loading capacity and the enhancement of the catalytic activity probably arise from the Coulombic interactions between the enzyme and MSPB. These findings provide a practical strategy for enhancement of enzyme activity and enzyme recycling by MSPB.

Optimizing the selective recognition of protein isoforms through tuning of nanoparticle hydrophobicity.

We demonstrate that ligand hydrophobicity can be used to increase affinity and selectivity of binding between monolayer-protected cationic gold nanoparticles and β-lactoglobulin protein isoforms containing two amino acid mutations.

Enhanced Electrostatic Discrimination of Proteins on Nanoparticle-Coated Surfaces.

Two β-lactoglobulin (BLG) isoforms, BLGA and BLGB, were used a test bed for the differentiation of proteins using electrostatics. In these studies, the BLGA and BLGB binding to a highly charged, cationic gold nanoparticle (GNP) modified surface was investigated by atomic force microscopy (AFM) and surface plasmon resonance (SPR) spectroscopy The binding affinity, and more importantly, the selectivity of this surface towards these two almost identical protein isoforms were both significantly increased on the cationic GNP surface array relative to the values measured with the same free cationic GNP in solution. While protein recognition is traditionally achieved almost exclusively via orientation dependent short-range interactions such as hydrogen bonds and hydrophobic interactions, our results show the potential of protein recognition platforms based on enhanced electrostatic interactions.

Heavy Metal Ions Removal by Nano-sized Spherical Polymer Brushes

Nano-sized spherical polymer brushes (SPBs) consisting of both a polystyrene (PS) core and a brush shell of poly(acrylic acid) (PAA), poly(N-acrylcysteamine) (PSH), or poly(N-acrylcysteamine-co-acrylic acid) (P(SH-co-AA)), were prepared by photo-emulsion polymerization. The core-shell structure was observed by dynamic light scattering and transmission electron microscopy. Due to the strengthened Donnan effect, the PAA brush can adsorb heavy metal ions. Effects of the contact time, thickness of PAA brush and pH value on the adsorption results were investigated. Due to the coordination between the mercapto groups and heavy metal ions as well as the electrostatic interactions, SPBs with mercapto groups are capable to remove heavy metal ions selectively from aqueous solutions. The order of adsorption capacity of the heavy metal ions by SPBs with mercapto groups is: Hg2+ ≈ Au3+ > Pb2+ > Cu2+ > Ni2+. The adsorbed heavy metal ions can be eluted from SPB by aqueous HCl solution, and the SPBs can be recovered. After three regenerations the recovered SPBs still maintain their adsorption capacity.

Antifouling performance of nano-sized spherical poly(N-hydroxyethyl acrylamide) brush

The biomedical applications of nanoparticles are still impeded by the non-specific adsorption of proteins, cells, or others biological species in vivo/in vitro. In this work, poly(N-hydroxyethyl acrylamide) was hired to modify a solid polymer core, polystyrene (PS) nanoparticles, via surface-initiated photo-emulsion polymerization to form nano-sized spherical poly(N-hydroxyethyl acrylamide) brush (PS@PHEAA). Its antifouling ability and stability were investigated by dynamic light scattering (DLS), turbidimetric titration, and isothermal titration calorimetry (ITC). The size of PS@PHEAA was constant as a function of pH, while slightly changed with ionic strength in single protein solution. ITC data confirmed that protein was slightly adsorbed on PS@PHEAA and the ionic strength influenced the adsorption. All characterizations demonstrated that PHEAA layer reduced the interaction between nanoparticles and proteins. Thus, these nanoparticles ideal candidates for future applications in the biomedical field.