Elena Piletska

Influence of Surface‐Imprinted Nanoparticles on Trypsin Activity

Here, the modulation of enzyme activity is presented by protein-imprinted nanoparticles produced using a solid-phase approach. Using trypsin as target, binding of the nanoparticles to the enzyme results in its inhibition or in stabilization, depending on the orientation of the immobilized enzyme used during imprinting.

Molecularly imprinted polymer cartridges coupled to high performance liquid chromatography (HPLC-UV) for simple and rapid analysis of fenthion in olive oil

A combination of molecular modelling and a screening of the library of non-imprinted polymers (NIPs) was used to identify acrylamide as a functional monomer with high affinity towards fenthion, organophosphate insecticide, which is frequently used in the treatment of olives. A good correlation was found between the screening tests and modelling of monomer-template interactions performed using a computational approach. Acrylamide-based molecularly imprinted polymer (MIP) and non-imprinted polymer (NIP) were thermally synthesised in dimethyl formamide (porogen) using ethylene glycol dimethacrylate as a cross-linker and 1,1-azo-bis (isobutyronitrile) as an initiator. The chemical and physical properties of the prepared polymers were characterised. The binding of fenthion by the polymers was studied using solvents with different polarities. The developed MIP showed a high selectivity towards fenthion, compared to other organophosphates (dimethoate, methidathion malalthion), and allowed extraction of fenthion from olive oil samples with a recovery rate of about 96%. The extraction of fenthion using MIPs was much more effective than traditional C18 reverse-phase solid phase extraction and allowed to achieve a low detection limit (LOD) (5 µg L(-1)).

Novel linear polymers able to inhibit bacterial quorum sensing.

Bacterial phenotypes, such as biofilm formation, antibiotic resistance and virulence expression, are associated with quorum sensing. Quorum sensing is a density-dependent regulatory system of gene expression controlled by specific signal molecules, such as N-acyl homoserine lactones (AHLs), produced and released by bacteria. This study reports the development of linear polymers capable to attenuate quorum sensing by adsorption of AHLs. Linear polymers were synthesized using MMA as backbone monomer and methacrylic acid and itaconic acid as functional monomers. Two different quorum sensing-controlled phenotypes, Vibrio fischeri bioluminescence and Aeromonas hydrophila biofilm formation, were evaluated to test the polymers efficiency. Results showed that both phenotypes were significantly affected by the polymers, with the itaconic acid-containing material being more effective than the methacrylic acid one. The polymer inhibitory effects were reverted by the addition of lactones, confirming attenuation of quorum sensing through sequestration of signal molecules. The polymers also showed no cytotoxicity when tested using a mammalian cell line.

A pseudo-ELISA based on molecularly imprinted nanoparticles for detection of gentamicin in real samples

The enzyme-linked immunosorbent assay (ELISA) is one of the most widely employed tests in diagnostics, and it relies on the use of antibodies to quantify the molecule of interest. Molecularly imprinted nanoparticles (nanoMIPs), thanks to their stability, cost efficiency and easy production, are a promising alternative to antibodies in assays and sensors. In this work, nanoMIPs have been produced by means of a solid-phase approach and employed for the detection of gentamicin in real samples. The produced nanoMIPs were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques. The determination of gentamicin in spiked milk was implemented through an assay similar to enzyme-linked immunosorbent assay, in which the nanoMIPs were used as a synthetic capture antibody (pseudo-ELISA). The detection of gentamicin was achieved in competitive binding experiments with a horseradish peroxidase–gentamicin conjugate. Gentamicin was determined in milk at clinically relevant concentrations with a mean accuracy of 94%. The cross-reactivity of such nanoparticles was investigated with streptomycin and ampicillin as control antibiotics, demonstrating excellent specificity.

Molecular modelling and synthesis of a polymer for the extraction of amiloride and triamterene from human urine

A new solid-phase extraction resin with high affinity towards amiloride and triamterene was developed with the assistance of computational modelling. Itaconic acid was selected as the best functional monomer capable of forming strong complexes with amiloride and triamterene. Conditions for selective solid-phase extraction of the diuretics from human urine and for their separation using a custom-designed adsorbent were optimised. It was shown that this adsorbent provided a high level of purification of the diuretics from human urine, which made their quantification using spectrophotometry possible.

Fluorescent sensor systems based on nanostructured polymeric membranes for selective recognition of Aflatoxin B1

Nanostructured polymeric membranes for selective recognition of aflatoxin B1 were synthesized in situ and used as highly sensitive recognition elements in the developed fluorescent sensor. Artificial binding sites capable of selective recognition of aflatoxin B1 were formed in the structure of the polymeric membranes using the method of molecular imprinting. A composition of molecularly imprinted polymer (MIP) membranes was optimized using the method of computational modeling. The MIP membranes were synthesized using the non-toxic close structural analogue of aflatoxin B1, ethyl-2-oxocyclopentanecarboxylate as a dummy template. The MIP membranes with the optimized composition demonstrated extremely high selectivity towards aflatoxin B1 (AFB1). Negligible binding of close structural analogues of AFB1 - aflatoxins B2 (AFB2), aflatoxin G2 (AFG2), and ochratoxin A (OTA) was demonstrated. Binding of AFB1 by the MIP membranes was investigated as a function of both type and concentration of the functional monomer in the initial monomer composition used for the membranes synthesis, as well as sample composition. The conditions of the solid-phase extraction of the mycotoxin using the MIP membrane as a stationary phase (pH, ionic strength, buffer concentration, volume of the solution, ratio between water and organic solvent, filtration rate) were optimized. The fluorescent sensor system based on the optimized MIP membranes provided a possibility of AFB1 detection within the range 14-500ngmL-1 demonstrating detection limit (3Ϭ) of 14ngmL-1. The developed technique was successfully applied for the analysis of model solutions and waste waters from bread-making plants.

Microplates with enhanced immobilization capabilities controlled by a magnetic field

New microtiter plates have been developed with immobilization capabilities controlled by a magnetic field. The innovation involves the use of disk-shaped inserts made of magnetic material, with apertures in the center of the disks. Ferromagnetic and paramagnetic nanoparticles and microparticles with immobilized biological or chemical reagents were quickly and effectively captured by the magnetic inserts. The immobilization of any ligand can be achieved in 2xa0min, its level directly proportional to the quantity of added particles. The proof of concept has been demonstrated by the immobilization of peroxidase, immunoglobulins and single-chain DNA. It was found that the immobilized proteins were capable of reacting with corresponding substrates and antigens. The internal opening of the disks allowed measurements of the reactions using standard microtiter plate readers. Magnetic microparticles and nanoparticles are currently used predominantly in separation. The technology described here allows their applicati...

Development of competitive ‘pseudo’-ELISA assay for measurement of cocaine and its metabolites using molecularly imprinted polymer nanoparticles

The analytical test-system for cocaine, benzoylecgonine and norcocaine was developed in the ELISA format using molecularly imprinted polymeric nanoparticles (nanoMIPs) as synthetic recognition elements that were produced using a solid-phase synthesis approach. The experimental conditions of the assay were optimized using a Box–Behnken experimental design protocol. The detection of free cocaine and its metabolites was performed using a competitive binding assay in the model samples and in blood plasma. There was no cross-reactivity of the developed assay towards paracetamol and caffeine. The developed assay had a picomolar limit of detection of cocaine (LOD = 4.24 pM), which was almost three orders of magnitude lower than the LOD expected from the commercial antibody-based ELISA (3.3 nM), and other attractive features of a new assay included a long shelf-life, lower economic cost and a short production time. Therefore, it is possible to state that nanoMIPs have the potential to become the recognition elements of choice for the development of a new generation of test-systems and sensors.

Solid phase extraction of α-tocopherol and other physiologically active components from sunflower oil using rationally designed polymers

A rationally designed polymer (RDP) capable of recognizing α-tocopherol and other minor components in sunflower oil has been produced. It is known that sunflower oil is a source of various physiologically active compounds. Unfortunately, they are present in very minor quantities which make their purification from the complex oil matrix problematic. An extraction method presented here was developed with particular attention to the selectivity, efficiency and precision of the extraction process. The methacrylic acid-based RDP in combination with the optimised purification method allowed the extraction of α-tocopherol with 94% recovery. The synthesised polymer was used successfully to extract α-tocopherol together with other essential minor components of sunflower oil without any pre-treatment step. According to GC/MS, the compounds ‘harvested’ from sunflower oil using the developed polymer included palmitic, oleic and linoleic acids, α-tocopherol, campesterol, stigmasterol, and β-sitosterol.

Computational design of molecularly imprinted polymer for direct detection of melamine in milk

ABSTRACT A novel protocol for use of molecularly imprinted polymer (MIP) in analysis of melamine is presented. Design of polymer for melamine has been achieved using a combination of computational techniques and laboratory trials, the former greatly reducing the duration of the latter. The compatibility and concerted effect of monomers and solvents were also investigated and discussed. Two novel open-source tools were presented which are the online polymer calculator from mipdatabase.com and the application of the Gromacs modelling suite to determine the ideal stoichiometric ratio between template and functional monomer. The MIP binding was characterised for several structural analogues at 1–100 μM concentrations. The use of divinylbenzene (DVB) as cross-linking polymer and itaconic acid as functional monomer allowed synthesis of MIP with imprint factor (IF) of 2.25 for melamine. This polymer was used in high-performance liquid chromatography (HPLC) for the rapid detection of melamine in spiked milk samples with an experimental run taking 7–8 min. This approach demonstrated the power of virtual tools in accelerated design of MIPs for practical applications.