Quantitative Detection of Trace Level Cloxacillin in Food Samples Using Magnetic Molecularly Imprinted Polymer Extraction and Surface-Enhanced Raman Spectroscopy Nanopillars.

There is an increasing demand for rapid, sensitive, and low cost analytical methods to routinely screen antibiotic residues in food products. Conventional detection of antibiotics involves sample preparation by liquid-liquid or solid-phase extraction, followed by analysis using liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis (CE), or gas chromatography (GC). The process is labor-intensive, time-consuming, and expensive. In this study, we developed a new analytical method that combines magnetic molecularly imprinted polymer (MMIP)-based sample preparation with surface-enhanced Raman spectroscopy (SERS)-based detection for quantitative analysis of cloxacillin in pig serum. MMIP microspheres were synthesized using a core-shell technique. The large loading capacity and high selectivity of the MMIP microspheres enabled efficient extraction of cloxacillin, while the magnetically susceptible characteristics greatly simplified sample handling procedures. Low cost and robust SERS substrates consisting of vertical gold capped silicon nanopillars were fabricated and employed for the detection of cloxacillin. Quantitative SERS was achieved by normalizing signal intensities using an internal standard. By coherently combining MMIP extraction and silicon nanopillar-based SERS biosensor, good sensitivity toward cloxacillin was achieved. The detection limit was 7.8 pmol. Cloxacillin recoveries from spiked pig plasma samples were found to be more than 80%.

The use of differential scanning fluorimetry in the rational design of plastic antibodies for protein targets.

The development of molecularly imprinted polymer nanoparticles (MIP-NPs), which specifically bind biomolecules, is of great interest in the area of biosensors, sample purification, therapeutic agents and biotechnology. Polymerisation techniques such as precipitation polymerisation, solid phase synthesis and core shell surface imprinting have allowed for significant improvements to be made in developing MIP-NPs which specifically recognise proteins. However, the development of MIP-NPs for protein templates (targets) still require lengthy optimisation and characterisation using different ratios of monomers in order to control their size, binding affinity and specificity. In this work we successfully demonstrated that differential scanning fluorimetry (DSF) can be used to rapidly determine the optimum imprinting conditions and monomer composition required for MIP-NP design and polymerisation. This is based on the stability of the protein template and shift in apparent melting points (Tm) upon interaction with different functional acrylic monomers. The method allows for the characterisation of molecularly imprinted nanoparticles (MIP-NPs) due to the observed differences in melting point profiles between, protein-MIP-NPs complexes, pre-polymerisation mixtures and non-imprinted nanoparticles (NIP-NPs) without the need for prior purification. The technique is simple, rapid and can be carried out on most quantitative polymerase chain reaction (qPCR) thermal cyclers which have the required filters for SYPRO

A multifunctional molecularly imprinted polymer-based biosensor for direct detection of doxycycline in food samples.

In this study, we developed a new type of multifunctional molecularly imprinted polymer (MIP) composite as an all-in-one biosensor for the low-cost, rapid and sensitive detection of doxycycline in pig plasma. The MIP composite consisted of a magnetic core for ease of manipulation, and a shell of fluorescent MIPs for selective recognition of doxycycline. By simply incorporating a small amount of fluorescent monomer (fluorescein-O-acrylate), the fluorescent MIP layer was successfully grafted onto the magnetic core via a surface imprinting technique. The resultant MIP composites showed significant doxycycline-dependent fluorescence quenching in an aqueous environment. Good linearity ranging from 0.2 to 6 µM was achieved, and the limit of detection was determined to be 117 nM. The biosensor also showed good selectivity towards doxycycline when compared to other common antibiotic residues. The multifunctional MIP composites were used to directly extract doxycycline from spiked pig plasma samples and quantify the antibiotics based on the quenched fluorescence signals. Recoveries of doxycycline were found in the range of 88-107%.

Detection of hemoglobin using hybrid molecularly imprinted polymers/carbon quantum dots-based nanobiosensor prepared from surfactant-free Pickering emulsion.

A simple fluorescent nanobiosensor based on molecularly imprinted polymers (MIPs) and carbon quantum dots (CQDs) was developed for hemoglobin (Hb) detection. The nanocomposites were synthesized by a novel one-pot surfactant-free Pickering emulsion method, in which imprinted cavities complementary to Hb were formed at the surface of the particles for target recognition, while CQDs were incorporated in the core as the fluorescence probe. We innovatively used the Hb template as emulsifier to help stabilize the emulsion droplets. The method eliminated the need of surfactant, which greatly simplified Pickering emulsion synthesis procedures, and significantly enhanced the fidelity of molecular imprinting. Moreover, the method provided an easy way to integrate fluorescent probes with MIPs in a single step. The nanobiosensor was utilized for determination of Hb via fluorescence quenching, and high selectivity and sensitivity were achieved. Under the optimized conditions, a linear range of 0.77-7.7 nM and a detection limit of 0.77 nM were obtained. The resulting nanocomposites were also successfully applied to detect Hb in the serum samples, which showed good recoveries ranging from 86.8% to 93.9%.

Dispersive solid-phase imprinting of proteins for the production of plastic antibodies.

We describe a novel dispersive solid-phase imprinting technique for the production of nano-sized molecularly imprinted polymers (nanoMIPs) as plastic antibodies. The template was immobilized on in-house synthesized magnetic microspheres instead of conventional glass beads. As a result, high-affinity and template-free MIPs were produced in higher yields.

Synthesis of Molecularly Imprinted Polymer Nanoparticles for α-Casein Detection Using Surface Plasmon Resonance as a Milk Allergen Sensor.

Food recalls due to undeclared allergens or contamination are costly to the food manufacturing industry worldwide. As the industry strives for better manufacturing efficiencies over a diverse range of food products, there is a need for the development of new analytical techniques to improve monitoring of the presence of unintended food allergens during the food manufacturing process. In particular, the monitoring of wash samples from cleaning in place systems (CIP), used in the cleaning of food processing equipment, would allow for the effective removal of allergen containing ingredients in between food batches. Casein proteins constitute the biggest group of proteins in milk and hence are the most common milk protein allergen in food ingredients. As such, these proteins could present an ideal analyte for cleaning validation. In this work, molecularly imprinted polymer nanoparticles (nanoMIPs) with high affinity toward bovine α-casein were synthesized using a solid-phase imprinting method. The nanoMIPs were then characterized and incorporated into label free surface plasmon resonance (SPR) based sensor. The nanoMIPs demonstrated good binding affinity and selectivity toward α-casein (KD ∼ 10 × 10-9 M). This simple affinity sensor demonstrated the quantitative detection of α-casein achieving a detection limit of 127 ± 97.6 ng mL-1 (0.127 ppm) which is far superior to existing commercially available ELISA kits. Recoveries from spiked CIP wastewater samples were within the acceptable range (87-120%). The reported sensor could allow food manufacturers to adequately monitor and manage food allergen risk in food processing environments while ensuring that the food produced is safe for the consumer.

Molecularly imprinted polymers for sample preparation and biosensing in food analysis: Progress and perspectives.

Molecularly imprinted polymers (MIPs) are biomimetics which can selectively bind to analytes of interest. One of the most interesting areas where MIPs have shown the biggest potential is food analysis. MIPs have found use as sorbents in sample preparation attributed to the high selectivity and high loading capacity. MIPs have been intensively employed in classical solid-phase extraction and solid-phase microextraction. More recently, MIPs have been combined with magnetic bead extraction, which greatly simplifies sample handling procedures. Studies have consistently shown that MIPs can effectively minimize complex food matrix effects, and improve recoveries and detection limits. In addition to sample preparation, MIPs have also been viewed as promising alternatives to bio-receptors due to the inherent molecular recognition abilities and the high stability in harsh chemical and physical conditions. MIPs have been utilized as receptors in biosensing platforms such as electrochemical, optical and mass biosensors to detect various analytes in food. In this review, we will discuss the current state-of-the-art of MIP synthesis and applications in the context of food analysis. We will highlight the imprinting methods which are applicable for imprinting food templates, summarize the recent progress in using MIPs for preparing and analysing food samples, and discuss the current limitations in the commercialisation of MIPs technology. Finally, future perspectives will be given.