Development in competitive immunoassay of a point-of-care testing for cotinine (COT) detection in urine†

We present a sensitive and selective lateral flow immunoassay (LFIA) for cotinine (COT), the primary metabolite of nicotine. COT is widely recognized as a superior biomarker to evaluate tobacco smoke exposure. The LFIA uses a competitive assay format where the COT–BSA capture competes with the target COT in urine samples for binding to the monoclonal antibody against COT (mAb–COT) conjugated with gold nanoparticles (mAb–COT–AuNPs). To improve the sensitivity and selectivity of the LFIA-COT, we focused on optimizing the diameter of AuNPs, the conjugation of mAb–COT, and the concentration of the COT–BSA capture. Our findings reveal that the utilization of 40 nm AuNPs in conjugation with a concentration of 4 mg mL⁻¹ of mAb–COT demonstrated significantly greater efficacy compared to LFAs utilizing 20 nm AuNPs. Under the optimal conditions, the LFIA-COT demonstrated sensitive detection of COT at a level of 150 ng mL⁻¹ within 15 min, as observed by the naked eye. It possesses a linear range of 25 to 200 ng mL⁻¹ of COT, with the limit of detection (LOD) of 11.94 ng mL⁻¹ in human urine samples when the color intensity is analyzed using ImageJ software. Our LFIA described here is simple and requires less time for COT detection. It can be used for the rapid and quantitative detection of COT in urine samples in clinical settings.