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Core–Shell Molecularly Imprinted Polymer Nanoparticles as Synthetic Antibodies in a Sandwich Fluoroimmunoassay for Trypsin Determination in Human Serum

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Sorbonne Universités, Université de Technologie de Compiègne, CNRS Enzyme and Cell Engineering Laboratory, Rue Roger Couttolenc, CS 60319, 60203 Compiègne Cedex, France
*E-mail: [email protected] (B. Tse Sum Bui).
*E-mail: [email protected] (K. Haupt).
Cite this: ACS Appl. Mater. Interfaces 2017, 9, 29, 24476–24483
Publication Date (Web):July 5, 2017
Copyright © 2017 American Chemical Society

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    We describe the application of a fluorescently labeled water-soluble core–shell molecularly imprinted polymer (MIP) for fluorescence immunoassay (FIA) to detect trypsin. p-Aminobenzamidine (PAB), a competitive inhibitor of trypsin, was immobilized in the wells of a microtiter plate enabling the capture of trypsin in an oriented position, thus maintaining its native conformation. Fluorescent MIP nanoparticles, which bound selectively to trypsin, were used for quantification. The MIP was prepared by a multistep solid-phase synthesis approach on glass beads functionalized with PAB, orientating all trypsin molecules in the same way. The core–MIP was first synthesized, using a thermoresponsive polymer based on N-isopropylacrylamide, so as to enable its facile liberation from the immobilized template by a simple temperature change. The shell, mainly composed of allylamine to introduce primary amino groups for postconjugation of fluorescein isothiocyanate (FITC), was grafted in situ on the core–MIP, whose binding cavities were still bound and protected by the immobilized trypsin. The resulting core–shell MIP was endowed with a homogeneous population of high-affinity binding sites, all having the same orientation. The MIP has no or little cross-reactivity with other serine proteases and unrelated proteins. Our MIP-based FIA system was successfully applied to detect low trypsin concentrations spiked into nondiluted human serum with a low limit of quantification of 50 pM, which indicates the significant potential of this assay for analytical and biomedical diagnosis applications.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b05844.

    • Details of the synthesis of the iniferter, diethylthiocarbamoylsulfanyl acetic acid, the characterization of core–shell polymers (size, zeta potential, molecular weight, and fluorescence analysis), activation and characterization of microplates, optimization of cytochrome c amount for use in the quantification of trypsin, and quantification of trypsin by using cytochrome c as substrate (PDF)

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