Rebinding Behavior Research Articles

Crucial Roles of Bond Rebinding in Rupture Behaviors of Single Molecular Bond at Ultralow Loading Rates

Single-molecule dynamic force spectroscopy (DFS) is a powerful tool for studying mechanical forces of molecular interactions. Recently, the important role of bond rebinding in DSF experiments was recognized, which intrigued mounting researches in this direction. In this work, we study how the bond rebinding influences the strength of single-molecular bonds using Brownian dynamics (BD) simulations and theoretical modeling. Our results show that bond rebinding significantly enhances the strength of single-molecular bond at ultralow loading rates. The rebinding behavior strongly depends on the loading stiffness, suggesting that the strength of single-molecular bond is not only dependent on its intrinsic property, but also the stiffness of loading device. By connecting our new model with conventional theories that did not consider the rebinding effect and are only applied to high loading rates, we propose a unified scheme to predict the rupture forces in a full range of loading rate in DSF experiments and simulations.

The imprinting induce‐fit model of specific rebinding of macromolecularly imprinted polymer microspheres

AbstractThe rebinding behavior of protein‐imprinted hydrogel is different from classical small molecularly imprinted polymer especially in protein aggregating state and interaction changeability between protein and imprinted sites. BSA‐imprinted calcium alginate‐hydroxy ethyl cellulose microspheres were prepared, and the rebinding behavior was studied. An “imprinting induce‐fit model” was proposed to describe the rebinding property of protein‐imprinted hydrogel. Three kinds of different interacting forms between protein and imprinted hydrogel were discovered by Scatchard analysis. Slogistic fit analysis of rebinding rate coefficient was carried out, and the imprinted hydrogel was found capable of promoting rebinding through induce‐fit behavior. Higher imprinting efficiency was found in microsphere samples with lower crosslinking density. Rebinding regression equation was established, and the rebinding quantity was computable with parameters including BSA aggregate concentration [Pn], dissociating rate (k1), and single molecule rebinding rate (k2). The experimental and calculated rebinding concentration were compared, and errors between −9.43% and 5.59% were found. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci 122:1847–1856, 2011

Preparation and characterization of molecularly imprinted microspheres for selective extraction of trace melamine from milk samples

We describe molecularly imprinted microspheres (MIMs) for the selective extraction of melamine from milk. The MIMs were made from melamine as the template molecule, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the linking agent. The MIMs were synthesized by suspension polymerization and characterized by rebinding experiments. They displayed high adsorption capacity, fast rebinding kinetics, and highly specific rebinding of melamine. The imprinting factor is 4.1. Scatchard analysis revealed a one-type rebinding behavior, the dissociation constant and maximum rebinding capacity being 37.59 g L−1 and 30.85 μmol g−1, respectively. The MIMs exhibited a 25% cross-reactivity towards atrazine, but less than 3.0% towards prometryn, clenbuterol and metronidazole. In addition, a MIM-based solid phase extraction (MISPE) column for melamine was prepared by packing MIMs into a common SPE cartridge. The MISPE extraction gave recoveries of 89.8 to 100.6% of melamine, with relative standard deviations of 5.9 to 7.5%. There was no significant loss of rebinding capacity after more than 60 repeated uses, thus demonstrating the high stability of the MISPE column. The MSPE column also was applied to the extraction of melamine from spiked liquid and powdered milk with satisfying accuracy and precision.

Pulling Genetic RNA out of Tobacco Mosaic Virus Using Single-Molecule Force Spectroscopy

RNA-coat protein interactions in intact tobacco mosaic virus have been investigated for the first time directly on the single-molecule level by pulling the genetic RNA step by step out of the helical groove formed by its protein coat. The effects of pulling speed and pH on RNA-protein interactions are presented. In addition, the rebinding behavior of the detached RNA with the protein coat is discussed. Our results demonstrate the possibility of studying nucleic acid-protein interactions in more complicated systems using AFM-based single-molecule force spectroscopy.

Novel bioactive scaffolds with fibronectin recognition nanosites based on molecular imprinting technology

AbstractBiomimetic materials for application in the field of tissue engineering are usually obtained through covalent bonding between the polymer backbone and the bioactive molecules. A totally new approach, proposed for the first time by our research group, for the creation of advanced synthetic support structures for cell adhesion and proliferation is represented by molecular imprinting (MI) technology. In this article, we describe the synthesis and characterization of molecularly imprinted polymers with recognition properties toward a fibronectin peptide sequence and their application as functionalization structures. Polymers, in the form of densely fused microgel particles, were obtained by precipitation polymerization. The imprinted particles showed good performance in terms of recognition capacity and quantitative rebinding; moreover, the epitope effect was observed, with the particles able to recognize and rebind not only the specific peptide sequence but also a larger fibronectin fragment. The cytotoxicity tests showed normal vitality in C2C12 myoblasts cultured in a medium that was put in contact with the imprinted particles. Therefore, imprinted particles were used to functionalize synthetic polymeric films by deposition on their surface. The deposition of the imprinted particles did not alter their specific recognition and rebinding behavior. The most remarkable result was obtained by the biological characterization: in fact, the functionalized materials appeared able to promote cell adhesion and proliferation. These results are very promising and suggest that MI can be used as an innovative functionalization technique to prepare bioactive scaffolds with an effective capacity for improving tissue regeneration. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Effect on rebinding behavior with different composition and structure of the dually imprinted alginate polymer microspheres using proteins and o/w emulsion drops as dual templates

AbstractIn this study, the dually imprinted alginate polymer microspheres are prepared using proteins and o/w emulsion drops as dual templates with different structure and composition such as the content of hydroxylethyl cellulose (HEC), crosslinking degree, and porous size, etc. The rebinding tests are carried on egg white albumin (EA) protein imprinted alginate polymer microspheres with interpenetrating networks (IPNs) under various conditions like ionic strength, temperature, protein concentration, and pH value. Results showed that the IPNs with alginate and HEC by covalent bond influence the rigidity and swelling properties. Porous structure formed by introducing emulsion drops into alginate hydrogel has effect on the permeability and specific surface area of the polymer microspheres. The variation rule of rebinding behavior according to different HEC content and pore diameters reveals macromolecular imprinting effect to be different with pure pore creating effect compared with the emulsion imprinting effect. Moreover, the macromolecular and emulsion dually imprinting‐rebinding tests verify the co‐existing macromolecular imprinting effect and pore creating effect of emulsion imprinting. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Molecularly imprinted solid-phase extraction for the determination of fenitrothion in tomatoes

Organophosphorus insecticides are widely employed in agriculture, and residues of them can remain after harvesting or storage. Pesticide residue control is an important task for ensuring food safety. Common chromatographic methods used in the determination of pesticide residues in food require clean-up and concentration steps prior to quantitation. While solid-phase extraction has been widely employed for this purpose, there is a need to improve selectivity. Due to their inherent biomimetic recognition systems, molecularly imprinted polymers (MIP) allow selectivity to be enhanced while keeping the costs of analysis low. In this work, a MIP that was designed to enable the selective extraction of fenitrothion (FNT) from tomatoes was synthesized using a noncovalent imprinting approach. The polymer was prepared using methacrylic acid as functional monomer and ethyleneglycol dimethacrylate as crosslinking monomer in dichloromethane (a porogenic solvent). The polymer was characterized by Fourier transform infrared spectroscopy, solid-state nuclear magnetic resonance (NMR), scanning electron microscopy (SEM), and nitrogen sorption porosimetry. The pore structure and the surface area were evaluated using the BET adsorption method. To characterize the batch rebinding behavior of the MIP, the adsorption isotherm was measured, allowing the total number of binding sites, the average binding affinity and the heterogeneity index to be established. A voltammetric method of quantifying FNT during the molecularly imprinted solid-phase extraction (MISPE) studies was developed. The polymer was placed in extraction cartridges which were then used to clean up and concentrate FNT in tomato samples prior to high-performance liquid chromatographic quantitation. The material presented a medium extraction efficiency of 59% (for analyses performed with three different cartridges on three days and a fortification level of 5.0 microg g(-1)) and selectivity when used in the preparation of tomato samples, and presented the advantage that the polymer could be reused several times after regeneration.

Grafting of Ion-Imprinted Polymers on the Surface of Silica Gel Particles through Covalently Surface-Bound Initiators: A Selective Sorbent for Uranyl Ion

A new ion imprinted polymer coated silica gel sorbent has been prepared using the radical "grafting from" polymerization method through surface-bound azo initiators for selective uranyl uptake. The introduction of azo initiator onto the silica surface was achieved by the reaction of surface amino groups with 4,4'-azobis(4-cyanopentanoic acid chloride). The grafting step was then carried out in a stirred solution of initiator-modified silica particles in the presence of uranyl ion and functional and cross-linking monomers. The prepared sorbent was characterized using FT-IR spectroscopy, scanning electron microscopy (SEM), elemental analysis (EA), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and BET adsorption isotherm analysis. The influence of the uranyl concentration, pH, and flow rate of solution on the grafted polymer affinity has been investigated. Maximum uptake of uranyl ion was observed at a pH 3.0. The rebinding behavior of the sorbent has been successfully described by the Langmuir-Freundlich isotherm. The dynamic column capacity of sorbent and enrichment factor for uranyl ion were 52.9 +/- 3.4 micromol g(-1) and 52, respectively. It was found that imprinting results in increased affinity of the sorbent toward uranyl ion over strong competitor metal ions such as Fe(III) and Th(IV). The sorbent was repeatedly used and regenerated for 3 months without any significant decrease in polymer binding affinities. Finally the sorbent was applied to the preconcentration and determination of uranyl ion in real water samples.

Shape selectivity versus functional group pre-organization in molecularly imprinted polymers

A systematic survey of related molecular probes differing in shape or functional group orientation was used to compare the effects of shape selectivity versus pre-organization of functional monomers on imprinting and rebinding performance of molecularly imprinted polymers (MIPs). These studies revealed that templates with two functional group interactions with the MIPs are influenced to a larger degree by shape selective interactions than templates with three functional group interactions. For example, with two functional group interactions, increasing side chain size of compounds 1– 5 increased selectivity 5-fold; while the same size change for compounds with three functional group interactions leads to a maximum 2-fold increase. Thus, the effects of shape selectivity and pre-organization of functional groups do not appear to work in concert with each other during the imprinting process or in the rebinding behavior. Furthermore, greater selectivity is generally found for templates with two functional groups, where the dominant mode of molecular recognition is shape selectivity. For example, the α value for the MIP elicited toward template compound 5 with two hydrogen bonding groups was 5–12-fold higher than equivalently shaped compounds 6– 8 that have three non-covalent binding interactions (Table 3). On the other hand, pre-organization of functional groups dominated the performance of MIPs elicited toward templates with three template–functional group interactions. This is observed in Tables 6–8, where compounds with identical positioning of three functional groups all show less than an order of magnitude change in α values despite changes in shape.

Is re-association in folded proteins a case of nonextensivity?

Pioneering experiments by Frauenfelder and collaborators have shown that the ligands of iron (CO, O 2) in heme proteins such as myoglobin and hemoglobin, when photo-dissociated at temperatures up to about T ∗ (for example, T ∗= 160 K for MbCO in glycerol-water) exhibit an anomalous (power-law, instead of exponential) rebinding behavior as a function of time. We show here that this behavior is consistent with nonextensive statistics that have been successfully applied to other physical phenomena.