Preparation of species-specific monoclonal antibody and development of fluorescence immunoassay based on fluorescence resonance energy transfer of carbon dots for accurate and sensitive detection of Alicyclobacillus acidoterrestris in apple juice

Abstract

The growth and metabolism of Alicyclobacillus acidoterrestris can lead to the spoilage of commercial fruit juice. Existing methods have some drawbacks such as complex sample pretreatment, skilled technician requirement, reduced sensitivity and specificity. Herein, a novel fluorescence immunoassay was developed using a monoclonal antibody (mAb) against A. acidoterrestris as the sensing element and carbon dots (CDs) as the signal response unit. The CDs can be quenched via fluorescence resonance energy transfer (FRET) by the oxidization product of p-phenylenediamine (PPD), a chromogenic substrate of horseradish peroxidase (HRP). This approach showed enhanced accuracy and sensitivity with relatively low limit of detection (LOD) of 6.16 × 102 CFU mL−1. Moreover, apple juice contaminated with 1 CFU mL−1 of A. acidoterrestris can be identified after 24 h enrichment. This fluorescence immunoassay could serve as a powerful tool for laboratory identification and on-site inspection of A. acidoterrestris, reducing the adverse effect on the quality of fruit juice.