In this study, a sensitive and rapid chemiluminescence (CL) approach was developed to determine the lowest concentration of urea in the luminescent system of bioluminescent earthworms based on the urea reaction with luciferin, a common substrate for bioluminescent earthworms. To this end, response surface methodology (RSM) was utilized through a Box–Behnken design (BBD) to optimize the key variables such as luciferin (2 × 10–4–2 × 10−2 M), urea (1 × 10–5–1 × 10–3 M), and the mixture of hydrogen peroxide (H2O2) with sodium hydroxide (NaOH) as oxidant (2 × 10–4–2 × 10−2 M) and also evaluate their interactive efects on the CL emission. The linear response of CL intensity was detected in the urea concentration ranging from 1 × 10–2 to 5 × 10−9 M (R2 = 0.9918). The detection limit under the optimum conditions was calculated 3.25× 10−10 M. This rapid and facile method required low amounts of reagent for the successful detection of urea. Based on the results, these methods can ofer an opportunity for the potential role of the bioluminescent earthworms in biologicahttps://doi.org/10.1007/s11696-022-02647-6l and non-biological analyses
