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

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

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

Design and fabrication of a smart sensor using in silico epitope mapping and electro-responsive imprinted polymer nanoparticles for determination of insulin levels in human plasma

A robust and highly specific sensor based on electroactive molecularly imprinted polymer nanoparticles (nanoMIP) was developed. The nanoMIP tagged with a redox probe, combines both recognition and reporting capabilities. The developed nanoMIP replaces enzyme-mediator pairs used in traditional biosensors thus, offering enhanced molecular recognition for insulin, improving performance in complex biological samples, and yielding high stability. Also, most of existing sensors show poor performance after storage. To improve costs of the logistics and avoid the need of cold storage in the chain supply, we developed an alternative to biorecognition system that relies on nanoMIP. NanoMIP were computationally designed using “in-silico” insulin epitope mapping and synthesized by solid phase polymerisation. The characterisation of the polymer nanoparticles was performed by transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier-transform Infrared (FT-IR) and surface plasmon resonance (SPR). The electrochemical sensor was developed by chemical immobilisation of the nanoMIP on screen printed platinum electrodes. The insulin sensor displayed satisfactory performances and reproducible results (RSD = 4.2%; n = 30) using differential pulse voltammetry (DPV) in the clinically relevant concentration range from 50 to 2000 pM. The developed nanoMIP offers the advantage of large number of specific recognition sites with tailored geometry, as the resultant, the sensor showed high sensitivity and selectivity to insulin with a limit of detection (LOD) of 26 and 81 fM in buffer and human plasma, respectively, confirming the practical application for point of care monitoring. Moreover, the nanoMIP showed adequate storage stability of 168 days, demonstrating the robustness of sensor for several rounds of insulin analysis

Sensor based on molecularly imprinted polymer membranes and smartphone for detection of Fusarium contamination in cereals

The combination of the generic mobile technology and inherent stability, versatility and cost-effectiveness of the synthetic receptors allows producing optical sensors for potentially any analyte of interest, and, therefore, to qualify as a platform technology for a fast routine analysis of a large number of contaminated samples. To support this statement, we present here a novel miniature sensor based on a combination of molecularly imprinted polymer (MIP) membranes and a smartphone, which could be used for the point-of-care detection of an important food contaminant, oestrogen-like toxin zearalenone associated with Fusarium contamination of cereals. The detection is based on registration of natural fluorescence of zearalenone using a digital smartphone camera after it binds to the sensor recognition element. The recorded image is further processed using a mobile application. It shows here a first example of the zearalenone-specific MIP membranes synthesised in situ using “dummy template”-based approach with cyclododecyl 2, 4-dihydroxybenzoate as the template and 1-allylpiperazine as a functional monomer. The novel smartphone sensor system based on optimized MIP membranes provides zearalenone detection in cereal samples within the range of 1–10 µg mL−1 demonstrating a detection limit of 1 µg mL−1 in a direct sensing mode. In order to reach the level of sensitivity required for practical application, a competitive sensing mode is also developed. It is based on application of a highly-fluorescent structural analogue of zearalenone (2-[(pyrene-l-carbonyl) amino]ethyl 2,4-dihydroxybenzoate) which is capable to compete with the target mycotoxin for the binding to zearalenone-selective sites in the membrane’s structure. The competitive mode increases 100 times the sensor’s sensitivity and allows detecting zearalenone at 10 ng mL−1. The linear dynamic range in this case comprised 10–100 ng mL−1. The sensor system is tested and found effective for zearalenone detection in maize, wheat and rye flour samples both spiked and naturally contaminated. The developed MIP membrane-based smartphone sensor system is an example of a novel, inexpensive tool for food quality analysis, which is portable and can be used for the “field” measurements and easily translated into the practice

Disposable paracetamol sensor based on electroactive molecularly imprinted polymer nanoparticles for plasma monitoring

A highly sensitive disposable electrochemical sensor for paracetamol was devised using electroactive molecularly imprinted polymers nanoparticles (nanoMIPs). NanoMIPs were prepared by solid-phase synthesis. Polymer composition included itaconic acid as a specific functional monomer and ferrocene as redox label, which confers electroactivity to the nanoparticles. NanoMIPs were characterised by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) and scanning electron microscopy (SEM). Sensors were fabricated by covalent attachment of nanoMIPs on screen-printed carbon electrodes and then employed for electrochemical determination of paracetamol using differential pulse voltammetry (DPV). The sensor was successfully evaluated in spiked human plasma with recoveries at 94–108 % and presenting a sensitivity of 6.18 ± 0.22 μA mM−1. The limit of detection and limit of quantification for the sensor were found to be 50 μM and 167 μM, respectively, in a linear concentration range between 0.1 and 1 mM. High selectivity was demonstrated, with no interference found in the presence of caffeine, procainamide or ethyl 4-aminobenzoate. The sensors exhibited high reproducibility (RSD, 4.8 %), fast response time (∼8 s) and acceptable shelf life (90 days), confirming its suitability for point of care diagnostic applications

Integration of smart nanomaterials for highly selective disposable sensors and their forensic applications in amphetamine determination

Screening drugs on the street and biological samples pose a challenge to law enforcement agencies due to existing detection methods and instrument limitations. Herein we present a graphene-assisted molecularly imprinted polymer nanoparticle-based sensor for amphetamine. These nanoparticles are electroactive by incorporating ferrocene in their structure. These particles act as specific actuators in electrochemical sensors, and the presence of a ferrocene redox probe embedded in the structure allows the detection of non-electroactive amphetamine. In a control approach, nanoparticles were covalently immobilised onto electrochemical sensors by drop-casting using silanes. Alternatively, nanoparticles were immobilised employing 3D printing and a graphene ink composite. The electrochemical performance of both approaches was evaluated. As a result, 3D printed nanoMIPs/graphene sensors displayed the highest selectivity in spiked human plasma, with sensitivity at 73 nA nM−1, LOD of 68 nM (RSD 2.4%) when compared to the silane drop cast electrodes. The main advantage of the optimised 3D printing technology is that it allows quantitative determination of amphetamine, a non-electroactive drug, challenging to detect with conventional electrochemical sensors. In addition, the cost-efficient 3D printing method makes these sensors easy to manufacture, leading to robust, highly selective and sensitive sensors. As proof of concept, sensors were evaluated on the street specimens and clinically relevant samples and successfully validated using UPLC-MS

Detecting and targeting senescent cells using molecularly imprinted nanoparticles

The progressive accumulation of senescent cells in tissues in response to damage importantly contributes to pathophysiological conditions such as fibrosis, diabetes, cancer, Alzheimer's and ageing. Consistent with this, eliminating senescent cells prolongs the lifespan and healthspan in animals and ameliorates certain diseases. Detecting and clearing senescent cells from human tissues could therefore have a significant diagnostic and prognostic impact. However, identifying senescent cells in vivo has proven to be complex. To address this, we characterized and validated a panel of novel membrane markers of senescence. Here, we show the application of molecularly imprinted nanoparticles (nanoMIPs) against an extracellular epitope of one of these markers, B2M, to detect senescent cells in vitro and in vivo. We show that nanoMIPs do not elicit toxic responses in the cells or in mice and successfully recognize old animals, which have a higher proportion of senescent cells in their organs. Importantly, nanoMIPs loaded with drugs can specifically kill senescent cells. Our results provide a proof-of-principle assessment of specific and safe nanotechnology-based approaches for senescent cell detection and clearance with potential clinical relevance