Direct and ultrasensitive optofluidic-based immunosensing assay of aflatoxin M1 in dairy products using organic solvent extraction

Aflatoxin M₁ (AFM₁), a highly toxic secondary metabolite, is present in a wide range of dairy products. In this study, we designed a simple, low-cost, reusable, and easy-to-operate immunosensing method for ultrasensitive detection of AFM₁ in dairy products by using a portable evanescent wave-based optofluidic biosensing platform (EOBP). The developed method provides the minimum detection limit of 5 ng/L, which is below the most restrictive standard imposed by the current regulations for AFM₁ in dairy products. The effect of several organic solvents, such as methanol, acetone, and acetonitrile, on the binding reaction of antibody-antigen in heterogeneous and homogeneous solutions was evaluated. Although the effect of organic solvents on the homogeneous binding reaction between antibody and antigen is more significant than that of heterogeneous binding reaction between antibody in solution and antigen immobilized onto the sensor surface, the fluorescence signal detected by EOBP is linearly dependent on AFM₁ concentration. Therefore, AFM₁ can be directly quantified even if the samples contain a certain organic solvent concentration. The robustness and stability of AFM₁-ovalbumin conjugate allow the regeneration of modified biosensor surface for more than 200 times, thereby achieving a cost-effective and reliable AFM₁ determination. The proposed method provides a rapid, ultrasensitive, and reliable AFM₁ determination in dairy products without complicated sample pretreatment process.