A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop assays similar to the enzyme-linked immunosorbent assay (ELISA) is presented here for the first time. NanoMIPs were synthesized by a solid-phase approach with an immobilized vancomycin (template) and characterized using Biacore 3000, dynamic light scattering, and electron microscopy. Immobilization, blocking, and washing conditions were optimized in microplate format. The detection of vancomycin was achieved in competitive binding experiments with a horseradish peroxidase-vancomycin conjugate. The assay was capable of measuring vancomycin in buffer and in blood plasma within the range of 0.001-70 nM with a detection limit of 0.0025 nM (2.5 pM). The sensitivity of the assay was 3 orders of magnitude better than a previously described ELISA based on antibodies. In these experiments, nanoMIPs have shown high affinity and minimal interference from blood plasma components. Immobilized nanoMIPs were stored for 1 month at room temperature without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELISA.

We report the development of an automated chemical reactor for solid-phase synthesis of MIP NPs in water. Operational parameters are under computer control, requiring minimal operator intervention. In this study, "ready for use" MIP NPs with sub-nanomolar affinity are prepared against pepsin A, trypsin and α-amylase in only 4 hours.The extent of research into biomedical and diagnostic nanotechnology is impressive, 1 despite currently very few examples of biomedical nanomaterials have been approved by the FDA. 2 Apart from the potential toxicity, the usage of nanomaterials in therapy and diagnostics finds a strong opponent in the well-established leadership of "safer" natural antibodies. 3 Industrial manufacturing of antibodies, however, is logistically complex and expensive, especially for applications in therapy. 4 Potential immunogenic adverse reactions, low stability and poor performance in non-physiological conditions restrict the application of antibodies to specific niche areas and shorten their shelf-life. 5,6 Finally, natural antibodies can be difficult to integrate with assays and sensors. 6,7 A possible alternative are MIP NPs, which share key characteristics with antibodies such as aqueous solubility, size, affinity and selectivity for the target. Additionally, solutions of MIP NPs can be handled similarly to solutions of antibodies, with the advantage of stability and robustness. 8 Moreover, operational parameters for producing MIP NPs can be carefully controlled. 9 There is, however, lack of a generic protocol for the synthesis of MIP NPs with specific characteristics, 8 which restricts their practical applications, despite recent reports of biological activity 10 and in vivo detoxification 11 hint at a great potential of MIP NPs in the nanomedicine arena.With the aim to provide a method that is scalable, reproducible and controlled, we have focused our efforts on the development of synthetic strategies for producing highperformance MIP NPs. The most suitable protocol should in our view include affinity purification with immobilised template to remove monomers/low-affinity NPs from the product. 12 Even better results would be obtained if the immobilised template is used also in polymerisation, as this will ensure high-binding site affinity and prevent the contamination 13 This approach would also add advantages of easy automation and the potential for scaling-up production.Recently we have reported the first successful example of solid-phase synthesis of MIP NPs using living-polymerisation chemistry, performed in organic solvent under UV irradiationconditions which are favourable for the imprinting of small molecules. 14 Imprinting of highmolecular weight targets such as proteins, polysaccharides and DNA, however, would require an aqueous environment to preserve their structure during polymerisation. 15Here we report the development of a reactor for MIP NPs preparation using chemical polymerisation performed in specifically mild aqueous conditions (monomer concentration: 6.5 mM) for p...

The synthesis of core-shell molecularly imprinted polymer nanoparticles (MIP NPs) has been performed using a novel solid-phase approach on immobilised templates. The same solid phase also acts as protective functionality for high affinity binding sites during subsequent derivatisation/shell formation. This procedure allows for the rapid synthesis, controlled separation and purification of high-affinity materials, with each production cycle taking just 2 hours. The aim of this approach is to synthesise uniformly-sized imprinted materials at the nanoscale which can be readily grafted with various polymers without affecting their affinity and specificity. For demonstration purposes we grafted anti-melamine MIP NPs with coatings which introduce the following surface characteristics: high polarity (PEG methacrylate); electro-activity (vinyl ferrocene); fluorescence (eosin acrylate); thiol groups (pentaerythritol tetrakis(3-mercaptopropionate)). The method has broad applicability and can be used to produce multifunctional imprinted nanoparticles with potential for further application in the biosensors, diagnostics and biomedical fields and as an alternative to natural receptors.

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