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

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

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

Development of a homogenous assay based on fluorescent imprinted nanoparticles for analysis of nitroaromatic compounds

Herein we describe the development of a homogeneous assay for the detection of 4-nitroaniline (4-NA) and 2,4-dinitroaniline (2,4-diNA). This assay relies on fluorescent molecularly imprinted nanoparticles (nanoMIPs) which, upon interaction with the target analytes, generate a reduction in fluorescence emission intensity (quenching). This is due to a responsive fluorescent monomer (N-2-propenyl-(5-dimethylamino)-1-naphthalene sulphonamide) employed in the manufacture of the nanoMIPs which, by virtue of the imprinting process, is capable of selective interaction with the target analyte, thus giving rise to a quenching effect. Selectivity experiments showed excellent recognition properties toward the target molecule. Under optimal conditions, the fluorescence intensity of these nanoMIPs decreased as the concentration of the imprinted analyte increased from 10 nM to 2.71 μM. A linear relation between the negative logarithm of 4-NA or 2,4-diNA concentrations and the fluorescence intensity for both nanosystems was found (R2 = 0.991 and R2 = 0.9895), with excellent sensitivity (limit of detection (LOD) = 7 and 6 nM, respectively). Furthermore, both nanosystems have been successfully applied for detection of 4-NA or 2,4-diNA in tap water, with recoveries between 90% to 100.6% and 92% to 100.3%, respectively. Thanks to the versatility of the imprinting process, this nanosystem holds the potential for further development of several optical sensors for many other compounds. [Figure not available: see fulltext.].</p

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