Solubility and size of polymer nanoparticles

The solubility of polymer nanoparticles is a complex phenomenon dependent on solvent–solute and solute–solute interactions. Contrary to phase separation in standard polymerization reactions, which is a well established research area, the relationship between the solubility of polymer nanoparticles and the resulting diameter of the nanoparticles has been largely overlooked. Herein we demonstrate that the absolute size of polymer nanoparticles can be predicted (and controlled) by varying the relevant parameters of the polymerization conditions that influence the solubility and Flory parameter, χs, p. The position of the spinodal, associated with a given χs, p equivalent and determined with a simple thermodynamic model, allows an absolute value, Δχspinodal, to be applied in predicting polymer dimensions. The hydrodynamic diameter of particles at the primarily observed fraction was found to be dependent on D (nm) = −74Δχspinodal + 367 nm, where Δχspinodal must be positive for successful separation. Variation with total polymer fraction over a limited range can also be observed to follow a trend of approximately D (nm) = 173 ln[(xN)2 10−36/Δχspinodal] − 193 nm, thus giving a more general description of polymerization. We also assert the importance of separating spinodal-character phase separation from binodal-character phase separation in polymer nanoparticle synthesis. To the best of our knowledge this is the first successful Flory–Huggins based thermodynamic model of polymer nanoparticles, and should provide a useful guide to predictive design of future nanomaterial

Development of a computationally-designed polymeric adsorbent specific for mycotoxin patulin.

Patulin is a toxic compound which is found predominantly in apples affected by mould rot. Since apples and apple-containing products are a popular food for the elderly, children and babies, the monitoring of the toxin is crucial. This paper describes a development of a computationally-designed polymeric adsorbent for the solid-phase extraction of patulin, which provides an effective clean-up of the food samples and allows the detection and accurate quantification of patulin levels present in apple juice using conventional chromatography methods. The developed bespoke polymer demonstrates a quantitative binding towards the patulin present in undiluted apple juice. The polymer is inexpensive and easy to mass-produce. The contributing factors to the function of the adsorbent is a combination of acidic and basic functional monomers producing a zwitterionic complex in the solution that formed stronger binding complexes with the patulin molecule. The protocols described in this paper provide a blueprint for the development of polymeric adsorbents for other toxins or different food matrices

Highly Efficient Abiotic Assay Formats for Methyl Parathion: Molecularly Imprinted Polymer Nanoparticle Assay as an Alternative to Enzyme-Linked Immunosorbent Assay

Enzyme-linked immunosorbent assay (ELISA) is a widely used standard method for sensitive detection of analytes of environmental, clinical, or biotechnological interest. However, ELISA has clear drawbacks related to the use of relatively unstable antibodies and enzyme conjugates and the need for several steps such as washing of nonbound conjugates and addition of dye reagents. Herein, we introduce a new completely abiotic assay where antibodies and enzymes are replaced with fluorescent molecularly imprinted polymer nanoparticles (nanoMIPs) and target-conjugated magnetic nanoparticles, which acted as both reporter probes and binding agents. The components of the molecularly imprinted polymer nanoparticle assay (MINA) are assembled in microtiter plates fitted with magnetic inserts. We have compared the performance of a new magnetic assay with molecularly imprinted polymer (MIP)-based ELISA for the detection of methyl parathion (MP). Both assays have shown high sensitivity toward allowing detection of MP at picomolar concentrations without any cross-reactivity against chlorpyriphos and fenthion. The fully abiotic assays were also proven to detect analyte in real samples such as tap water and milk. Unlike ELISA-based systems, the novel assay required no washing steps or addition of enzyme substrates, making it more user-friendly and suitable for high throughput screening

A magnetic molecularly imprinted nanoparticle assay (MINA) for detection of pepsin

A fully abiotic Molecularly Imprinted Nanoparticle Assay (MINA) is developed for detection of the model protein pepsin. The format of the pepsin assay is based on binding of fluorescent pepsin-specific molecularly imprinted polymer nanoparticles (nanoMIPs) to the magnetic pepsin nanoparticles (MPN) immobilised on magnetic microtiter plate inserts. Competition between free pepsin and immobilised pepsin results in a decrease in nanoMIPs binding to the magnetic inserts, resulting in an increase in fluorescence. Pepsin-specific are prepared using a solid phase synthesis protocol. In order to increase the sensitivity of MINA, two labelling approaches are performed. The first approach uses a fluorescein acrylate included in the monomeric mixture during polymerisation, and the second approach is based on a post-imprinting modification of nanoparticles using the commercial fluorophore AlexaFluor® 647 NHS ester. It is observed that upon increase of free pepsin concentration from 1 μM to 100 μM, fluorescence is increased by 68%. No cross-reactivity in the presence of non-specific protein trypsin is detected. The results show that AlexaFluor-labelled nanoMIPs demonstrate higher performance towards pepsin than fluorescein-labelled nanoMIPs. The novel assay reduces the time and cost of analysis and does not uses any antibodies, eliminating the need for animal-derived reagents. The portfolio of the optimised protocols can potentially be applied for the detection of any other proteins of clinical and environmental interest

Nanoparticle-induced enhancement of cholinesterase activity in the presence of malathion: A potential nerve agent therapeutic

Organophosphate nerve agents are associated with assassination, terrorism and chemical warfare, but there has been slow progress in developing a broad-spectrum response to poisoning. For some nerve agents the oxime component of the therapy may not be effective, limiting the effectiveness of emergency treatment that is desperately needed. An alternative therapy may be possible based on accelerating enzyme (acetylcholinesterase) catalysis in unaffected adjacent enzymes. Herein we demonstrate a restoration of acetylcholinesterase activity in malathion-inhibited cell membrane preparations by the administration of functional nanoparticles. The molecularly imprinted polymer nanoparticles were designed to bind selectively to designated enzyme epitopes. Enzyme activity of membrane-bound acetylcholinesterase was measured in the presence of the organophosphate malathion and the selected nanoparticles. Enzymatic acceleration of the cholinesterase was observed at 162 ± 17 % the rate of erythrocyte ghosts without bound nanoparticles. This may restore sufficient acetylcholine hydrolysis to mitigate the effects of poisoning, offsetting the acetylcholine accumulation resulting from enzyme inhibition

Application of molecularly imprinted polymer nanoparticles for degradation of the bacterial autoinducer N-hexanoyl homoserine lactone.

A novel bacterial quorum quenching system is presented. For the first time the degradation of N-l-hexanoyl homoserine lactone (C6-AHL), a Gram-negative quorum sensing autoinducer, has been enhanced using molecularly imprinted nanoparticles (MIP NPs) which were prepared using transition state analogue of the γ-lactone ring hydrolysis as template

Generic sensor platform based on electro-responsive molecularly imprinted polymer nanoparticles (e-NanoMIPs)

The present research describes the design of robust electrochemical sensors based on electro-responsive molecularly imprinted polymer nanoparticles (e-MIPs). The e-MIPs, tagged with a redox probe, combine both recognition and reporting functions. This system replaces enzyme-mediator pairs used in traditional biosensors. The analyte recognition process relies on the generic actuation phenomenon when the polymer conformation of e-MIPs is changing in response to the presence of the template analyte. The analyte concentration is measured using voltammetric methods. In an exemplification of this technology, electrochemical sensors were developed for the determination of concentrations of trypsin, glucose, paracetamol, C4-homoserine lactone, and THC. The present technology allows for the possibility of producing generic, inexpensive, and robust disposable sensors for clinical, environmental, and forensic applications

Size of Heparin-Imprinted Nanoparticles Reflects the Matched Interactions with the Target Molecule.

It has been shown that the faradic current at an electrode grafted with molecularly imprinted polymer (MIP) is sensitive to the specific target molecule used as the template. This phenomenon is applicable to sensors with very high selectivity, but the sensing mechanism is still a black box. We investigated the size sensitivity of nanoparticles of molecularly imprinted polymers (MIP-NPs) to a specific interaction for determination of the mechanism of the gate effect and its feasibility for new applications. Nanoparticles of poly(methacryloxy ethyl trimethylammonium chloride-co-acrylamide-co-methylenebisacrylamide) imprinted with heparin immobilized on glass beads were synthesized. The diameter of the MIP-NPs of heparin was increased by the presence of the heparin template but was insensitive to chondroitin sulfate C (CSC), the analogue of heparin. The high selectivity of the MIP-NPs was consistent with the selectivity of electrodes grafted with a heparin-imprinted polymer in our previous studies. The quartz crystal microbalance probes immobilizing heparin or CSC were sensitive to MIP-NPs, which indicates that the binding ability of MIP-NP does not discriminate between the template and other glycosaminoglycans. These results indicate that the size of the MIP-NP is sensitive to the matched binding with the template through the imprinted cavity

New protocol for optimisation of polymer composition for imprinting of peptides and proteins

We present here a novel screening tool for optimisation of polymerisation mixtures used in imprinting of peptides and proteins. To facilitate rapid synthesis and screening of a combinatorial library of polymers the solid-phase synthesis method developed by Piletsky and co-workers was scaled down to 50 mg of template-immobilised solid phase, allowing a single well of a 96-well microplate to function as an individual reaction vessel. In this way, 32 different polymer compositions containing N-isopropylacrylamide, acrylic acid, N-(3-aminopropyl)methacrylamide hydrochloride, and N-tert-butylacrylamide, were tested in imprinting of three peptides and three proteins. Utilising filtration microplates has allowed the elution and washing steps to be performed in a similar manner to the large-scale synthesis, whilst incorporation of a fluorescent monomer (N-fluoresceinylacrylamide) made it possible to analyse the binding of synthesised polymer nanoparticles to the solid phase with immobilised templates under different washing conditions. The experiment has proven that the variations in monomer compositions had an effect on the yield and affinity of synthesised molecularly imprinted polymers for the peptides, but not for the proteins. Imprinting in this way presents an ideal method for performing small-scale syntheses for testing polymerisation mixtures, as information regarding the molecularly imprinted polymers affinity can be assessed as part of the elution process, without a need for time-consuming analysis such as quartz crystal microbalance or surface plasmon resonance

Modulation of EGFR Activity by Molecularly Imprinted Polymer Nanoparticles Targeting Intracellular Epitopes

In recent years, molecularly imprinted polymer nanoparticles (nanoMIPs) have proven to be an attractive alternative to antibodies in diagnostic and therapeutic applications. However, several key questions remain: how suitable are intracellular epitopes as targets for nanoMIP binding? And to what extent can protein function be modulated via targeting specific epitopes? To investigate this, three extracellular and three intracellular epitopes of epidermal growth factor receptor (EGFR) were used as templates for the synthesis of nanoMIPs which were then used to treat cancer cells with different expression levels of EGFR. It was observed that nanoMIPs imprinted with epitopes from the intracellular kinase domain and the extracellular ligand binding domain of EGFR caused cells to form large foci of EGFR sequestered away from the cell surface, caused a reduction in autophosphorylation, and demonstrated effects on cell viability. Collectively, this suggests that intracellular domain-targeting nanoMIPs can be a potential new tool for cancer therapy