Development of luminol-N-hydroxyphthalimide chemiluminescence system for highly selective and sensitive detection of superoxide dismutase, uric acid and Co²⁺

N-hydroxyphthalimide (NHPI), a well known reagent in organic synthesis and biochemical applications, has been developed as a stable and efficient chemiluminescence coreactant for the first time. It reacts with luminol much faster than N-hydroxysuccinimide, eliminating the need of a prereaction coil used in N-hydroxysuccinimide system. Without using prereaction coil, the chemiluminescence peak intensities of luminol-NHPI system are about 102 and 26 times greater than that of luminol-N-hydroxysuccinimide system and classical luminol-hydrogen peroxide system, respectively. The luminol-NHPI system achieves the highly sensitive detection of luminol (LOD = 70 pM) and NHPI (LOD = 910 nM). Based on their excellent quenching efficiencies, superoxide dismutase and uric acid are sensitively detected with LODs of 3 ng/mL and 10 pM, respectively. Co²⁺ is also detected a LOD of 30 pM by its remarkable enhancing effect. Noteworthily, our method is at least 4 orders of magnitude more sensitive than previously reported uric acid detection methods, and can detect uric acid in human urine and Co²⁺ in tap and lake water real samples with excellent recoveries in the range of 96.35–102.70%. This luminol-NHPI system can be an important candidate for biochemical, clinical and environmental analysis.