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Sensitive determination of urea in luciferin chemiluminescence system using an experimental design

Afsaneh Nemati, Mohammad Javad Chaichi, Saman Hosseinkhani, Moslem Mansour Lakouraj, and Bagher Seyedalipour

Department of Analytical Chemistry, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran



Received: 16 July 2022  Accepted: 19 December 2022


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 effects 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 offer an opportunity for the potential role of the bioluminescent earthworms in biological and non-biological analyses.

Graphical abstract

Keywords: Bioluminescent earthworms; Box–Behnken design; Chemiluminescence; Luciferin; Urea

Full paper is available at

DOI: 10.1007/s11696-022-02647-6


Chemical Papers 77 (5) 2571–2580 (2023)

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