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Direct Replacement of Antibodies with Molecularly Imprinted Polymer Nanoparticles in ELISA—Development of a Novel Assay for Vancomycin

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Cranfield Health, Vincent Building, Cranfield University, Cranfield, Bedfordshire, MK43 0AL, U.K.
*E-mail: [email protected]. Tel.: +44 1234 758322. Fax: +44 1234 758380.
Cite this: Anal. Chem. 2013, 85, 17, 8462–8468
Publication Date (Web):August 15, 2013
Copyright © 2013 American Chemical Society

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    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.

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    Figures showing the Biacore testing to estimate dissociation constant of nanoMIPs, the calibration curves of HRP–V and HRP for dilutions of TMB reagent, and the results of experiments carried out to optimize washing buffer and quantity of nanoparticles per well are supplied as Supporting Information. This material is available free of charge via the Internet at

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