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.

A tri-signal ultrasensitive colorimetric/electrochemical detection of ovomucoid (OM) was developed. Herein, copper oxide nanoparticles (CuO NPs) were prepared, which exhibit excellent enzyme-like activity (peroxidase-like and laccase-like) and electrochemical activity. CuO@3-APBA nanoparticles (CuO@3-APBA NPs) were prepared by the coordinating Cu with the amino group on 3-aminophenobenic boric acid (3-APBA) in CuO NPs. 3-APBA of CuO@3-APBA can react with diol structure on sugar chain of OM under alkaline conditions. Then, a tri-signal ultrasensitive biosensing platform for OM was established based on the catalytic activity of CuO@3-APBA nanozyme. For the first signal, CuO@3-APBA can catalyze oxidation of TMB to turn the solution from colorless to blue in the presence of H₂O₂ (absorbance at 652 nm). For the second signal, CuO@3-APBA can catalyze the oxidation of substrates (2, 4-dichlorophenol and 4-aminoantipyrine) and turn  the solution from colorless to  pink (absorbance at 510 nm). For the third signal, electrochemical oxidation peak of copper ion from Cu⁺ to Cu²⁺ of CuO@3-APBA was recorded by differential pulse voltammetry, which was used to determine the OM. The sensing platform exhibited a wide linear range (0.0000316-100 ng/mL) with a low detection limit (0.0105 pg/mL), as well as showed advantages, such as satisfactory reproducibility, good stability, and excellent selectivity. The assay has the potential applications for ultrasensitive detection of allergen in foods.

Sensitive detection of Staphylococcus aureus enterotoxin B (SEB) is of importance for preventing food poisoning from threatening human health. In this work, an electrochemical and colorimetric dual-signal detection assay of SEB was developed. The probe (Ab2/AuPt@Fe-N-C) was bound to SEB captured by Ab1, where the Ab2/AuPt@Fe-N-C triggered methylene blue degradation and resulted in the decrease of electrochemical signal. Furthermore, the probe catalyzed the oxidation of 3, 3’, 5, 5’-tetramethyl biphenyl to generate a colorimetric absorbance at 652 nm. Once the target was captured and formed a sandwich-like complex, the color changed from colorless to blue. SEB detection by colorimetric and electrochemical methods showed a linear relationship in the concentration ranges of 0.0002–10.0000 and 0.0005–10.0000 ng/mL, with limits of detection of 0.0667 and 0.1670 pg/mL, respectively. The dual-signal biosensor was successfully used to detect SEB in milk and water samples, which has great potential in toxin detection in food and the environment.

Norovirus (NoV) is regarded as one of the most common causes of foodborne diarrhea in the world. It is urgent to identify the pathogenic microorganism of the diarrhea in short time. In this work, we developed an electrochemical and colorimetric dual-mode detection for NoV based on the excellent dual catalytic properties of copper peroxide/COF-NH₂ nanocomposite (CuO₂@COF-NH₂). For the colorimetric detection, NoV can be directly detected by the naked eye based on CuO₂@COF-NH₂ as a laccase-like nonazyme using “peptide-NoV-antibody” recognition mode. The colorimetric assay displayed a wide and quality linear detection range from 1 copy/mL to 5000 copies/mL of NoV with a low limit of detection (LOD) of 0.125 copy/mL. For the electrochemical detection of NoV, CuO₂@COF-NH₂ showed an oxidation peak of copper ion from Cu⁺ to Cu²⁺ using “peptide-NoV-antibody” recognition mode. The electrochemical assay showed a linear detection range was 1‒5000 copies/mL with a LOD of 0.152 copy/mL. It’s worthy to note that this assay does not need other electrical signal molecule, which provide the stable and sensitive electrochemial detection for NoV. The electrochemical and colorimetric dual-mode detection was used to detect NoV in foods and faceal samples, which has the potential for improving food safety and diagnosing of NoV-infected diarrhea.