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A magnetic molecularly imprinted nanoparticle assay (MINA) for detection of pepsin

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posted on 2022-01-13, 15:38 authored by Y García, J Czulak, ED Pereira, SA Piletsky, E Piletska
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.

History

Citation

Reactive and Functional Polymers Volume 170, January 2022, 105133

Author affiliation

School of Chemistry

Version

  • AM (Accepted Manuscript)

Published in

Reactive and Functional Polymers

Volume

170

Pagination

105133

Publisher

Elsevier BV

issn

1381-5148

Acceptance date

2021-11-26

Copyright date

2021

Available date

2023-06-10

Language

en

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