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Open Access

A novel fluorescence sensor for milk clotting enzyme chymosin using peptide as substrate and covalent organic framework nanosheet as fluorescence quencher

Xiaokang LuaTianle QiaLinjiang GuoaLin XiaoaHanbin XuaGuobao NingaHui Zhaob( )Canpeng Lia( )
School of Chemical Science and Technology, Yunnan University, Kunming 650091, China
Laboratory for Conservation and Utilization of Bio-Resource, Yunnan University, Kunming 650091, China

Peer review under responsibility of Tsinghua University Press.

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HIGHLIGHTS

• A simple fluorescence assay for chymosin detection was developed.

• The assay was constructed using the synthesized peptide as a substrate.

• Covalent organic framework nanosheet was used as a fluorescence quencher.

• Chymosin in commercial rennet product was successfully determined using the assay

• The assay showed good selectivity, excellent stability, and satisfactory reproducibility.

Graphical Abstract

Abstract

Chymosin is one of the critical enzymes in cheese making. Herein, we proposed a novel fluorometric assay for chymosin determination. Firstly, covalent organic frameworks (COF) were synthesized and exfoliated to 2-dimensional COF nanosheets (COF NS) by ultrasound treatment. Gold nanoparticles (Au NPs) were loaded with COF NS to prepare AuNPs/COF NS (Au@COF NS). Secondly, rhodamine B (RhB) modified substrate peptide (Pep) for chymosin was linked with Au@COF NS to construct a Pep-Au@COF NS nanocomposite. For the sensing principle, fluorescence of RhB was quenched by Au@COF NS and the fluorescence intensity was weak due to the fluorescence resonance energy transfer between COF NS and RhB of Pep. However, in the presence of chymosin, the RhB was released by specific cleavage of the substrate peptide by chymosin and resulted in the recovery of fluorescence. The increased fluorescence intensity was proportional to the increase of chymosin concentration and thus a "turn on" fluorescent sensor for chymosin was constructed. The sensor showed a linear range in the concentration of 0.05-60.00 μg/mL for the detection of chymosin with a detection limit of 20 ng/mL. The sensor was used to quantify chymosin in rennet product with good selectivity, which has the potential applications in cheese manufacturing.

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Food Science and Human Wellness
Pages 3606-3613
Cite this article:
Lu X, Qi T, Guo L, et al. A novel fluorescence sensor for milk clotting enzyme chymosin using peptide as substrate and covalent organic framework nanosheet as fluorescence quencher. Food Science and Human Wellness, 2024, 13(6): 3606-3613. https://doi.org/10.26599/FSHW.2023.9250042

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Received: 21 February 2023
Revised: 19 March 2023
Accepted: 09 May 2023
Published: 18 December 2024
© 2024 Beijing Academy of Food Sciences. Publishing services by Tsinghua University Press.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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