Application of the bespoke solid-phase extraction protocol for extraction of physiologically-active compounds from vegetable oils

A possibility of harvesting the physiologically-active compounds from the vegetable oils using bespoke resin and optimised solid-phase extraction (SPE) purification method is demonstrated. The paper describes the application of SPE protocol, which was originally developed, using sunflower oil as model biomass, for extraction of the valuable compounds from sesame, wheat germ, palm, olive and soybean oils. As a result, the extraction of three free fatty acids (palmitic, oleic and linoleic), α-tocopherol and three phytosterols (campesterol, stigmasterol and β-sitosterol) from six vegetable oils have been demonstrated. The comparison between the published data on the amount of the analysed compounds in the corresponding vegetable oils and presented here extraction results confirm that the developed method allowed not only quantitatively extract the physiologically-active components from various vegetable oils without any pre-treatment but also was reproducible, cost-effective and ecologically-conscious as consumed smaller volumes of organic solvents than commonly used protocols

Evading and overcoming AAV neutralization in gene therapy

Adeno-associated virus (AAV)-derived viral vectors are a promising platform for the delivery of curative, life-changing therapies to a huge number of patients with monogenic disorders. There are currently over 250 clinical trials ongoing worldwide. However, for these therapies to benefit as many patients as possible, techniques must be developed to treat those with pre-existing immunity and to potentially allow re-administration of a dose in the future, should efficacy wane over time. This review discusses the current state and prospects of technologies to evade and overcome these immune responses and allow successful treatment of the greatest number of patients possible

Molecularly imprinted nanoparticles-based assay (MINA) - detection of leukotrienes and insulin.

A novel molecularly imprinted polymer nanoparticle-based assay (MINA) performed in magnetic microplates was developed as an improved high-quality alternative to existing antibody-based immunoassays. MINA is a generic technology that can be adapted for biomarker detection in biological samples. Herein, we demonstrate the applicability of the MINA assay for the detection of leukotrienes and insulin in biological samples. MINA, used in a competition format, has allowed the detection of LTE4 in urine in a concentration range from 0.45 to 364 pM, with a LOD of 0.73 pM. MINA, used in a competition format, has allowed the detection of insulin in plasma in a concentration range from 25 to 2500 pM, with a LOD of 27 pM. This assay has shown comparable performance for LTE4 and insulin detection to existing chromatographic techniques (LC-MS/MS) and immunoassays in clinically relevant concentrations. The main advantages of this assay are the efficient and low cost fabrication, preparation of synthetic binders without the use of animals, and fewer steps used in the assay protocol as compared to traditional immunoassays

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

New potentiometric sensor based on molecularly imprinted nanoparticles for cocaine detection

Here we present a potentiometric sensor for cocaine detection based on molecularly imprinted polymer nanoparticles (nanoMIPs) produced by the solid-phase imprinting method. The composition of polymers with high affinity for cocaine was optimised using molecular modelling. Four compositions were selected and polymers prepared using two protocols: chemical polymerisation in water and UV-initiated polymerisation in organic solvent. All synthesised nanoparticles had very good affinity to cocaine with dissociation constants between 0.6nM and 5.3nM. Imprinted polymers produced in organic solvent using acrylamide as a functional monomer demonstrated the highest yield and affinity, and so were selected for further sensor development. For this, nanoparticles were incorporated within a PVC matrix which was then used to prepare an ion-selective membrane integrated with a potentiometric transducer. It was demonstrated that the sensor was able to quantify cocaine in blood serum samples in the range of concentrations between 1nM and 1mM

A Protocol for the Computational Design of High Affinity Molecularly Imprinted Polymer Synthetic Receptors

Molecularly imprinted polymer (MIP) nanoparticles, commonly referred to as 'plastic antibodies' or synthetic receptors, are polymeric materials with strong affinity and selectivity for a particular chemical target. MIPs are regularly produced for use in sensors for monitoring food quality and environmental pollutants, and in the design of robust and affordable replacements for biological receptors, enzymes and antibodies in drug testing and assays. More recently the easy production of MIP nanoparticles has also permitted research relating to possible in vivo applications, primarily in drug delivery systems, toxin sequestration and pathogen inhibition. The strength of the interaction between the target and the polymer binding site is dependent on the particular monomers selected in synthesis of the MIP, and the relative concentrations of these in the pre-polymerization mixture. While computational approaches have been used to aid in MIP design previously, the methods adopted are often slow and simplistic, centring on observations of the template structure with a couple of functional monomers presumed to be appropriate. We present here an automated method of rapidly screening numerous functional monomers and effectively determining appropriate monomer ratios, while accounting for spatial discrimination in selection and dynamic parameters in optimization. Example are then given of effect MIP synthesis resulting from the protocol, and the benefits of this approach over competing methods are discussed

DEVELOPMENT OF A SMARTPHONE-BASED BIOMIMETIC SENSOR FOR AFLATOXIN B1 DETECTION USING MOLECULARLY IMPRINTED POLYMER MEMBRANES

A novel smartphone-based optical biomimetic sensor based on free-standing molecularly imprinted polymer (MIP) membranes was developed for rapid and sensitive point-of-care detection of aflatoxin B1. The developed MIP membranes were capable of selective recognition of the target analyte and, at the same time, of generation of a fluorimetric sensor response, which could be registered using the camera of a smartphone and analysed using image analysis. The developed system provides a possibility of synchronous detection of aflatoxin B1 in 96 channels. UV irradiation of aflatoxin B1, selectively bound by the MIP membranes from the analysed samples, initiated fluorescence of aflatoxin B1 with intensity directly proportional to its concentration. The composition of the MIP membranes used as a recognition element was optimised taking into account data of computational modelling. Two functional monomers (2-acrylamido-2-methyl-1-propansulfonic acid and acrylamide) were identified as optimal for the formation of aflatoxin B1-selective binding sites in the structure of the MIP membranes. Working characteristics of the smartphone-based sensor system were also estimated. The influence of pH and of buffer and NaCl concentrations on the smartphone based sensor responses were studied. High selectivity of the developed sensor system towards aflatoxin B1 was confirmed in experiments with close structural analogues of the target analyte - aflatoxin G2, and ochratoxin A. The detection limit for aflatoxin B1 using the smartphone-based sensor systems was found to be 20 ng mL-1 for the sensor based on MIP membranes synthesised with acrylamide as a functional monomer. The storage stability of the recognition elements of the developed sensors was estimated as one year when stored at 22°C. The possibility to detect the aflatoxin B1 in contaminated food samples was shown. The MIP-membrane-based sensor system provided a convenient point-of-care approach in food safety testin

Development of MIP sensor for monitoring propofol in clinical procedures

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Modulation of acetylcholinesterase activity using molecularly imprinted polymer nanoparticles

Modulation of enzyme activity allows for control over many biological pathways and while strategies for the pharmaceutical design of inhibitors are well established; methods for promoting activation, that is an increase in enzymatic activity, are not. Here we demonstrate an innovative epitope mapping technique using molecular imprinting to identify four surface epitopes of acetylcholinesterase (AChE). These identified epitopes were then used as targets for the synthesis of molecularly imprinted nanoparticles (nanoMIPs). The enzymatic activity of AChE was increased upon exposure to these nanoMIPs, with one particular identified epitope nanoMIP leading to an increase in activity of 47× compared to enzyme only. The impact of nanoMIPs on the inhibited enzyme is also explored, with AChE activity recovering from 11% (following exposure to an organophosphate) to 73% (following the addition of nanoMIPs). By stabilizing the conformation of the protein rather than targeting the active site, the allosteric nature of MIP-induced reactivation suggests a new way to promote enzyme activity, even under the presence of an inhibitor. This method of enzyme activation shows promise to treat enzyme deficiency diseases or in medical emergencies where an external agent affects protein function

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

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 DVB as cross-linking polymer and itaconic acid as functional monomer allowed synthesis of MIP with imprint factor for melamine IF=2.25. This polymer was used in HPLC for the rapid detection of melamine in spiked milk samples with an experimental run taking 7-8 minutes. This approach demonstrated the power of virtual tools in accelerated design of MIPs for practical applications