Bisubstrate multi-colorimetric assay based on the peroxidase-like activity of Cu2+-triethylamine complex for copper ion detection

A bisubstrate colorimetric system was developed for rapid and multi-color assay of copper ion (Cu2+). Cu2+-triethylamine (Cu2+-TEA) complex with the peroxidase-like activity can catalyze the 3,3′,5,5′-tetramethylbenzidine (TMB) to produce oxidized TMB (TMB+, color in blue) and dopamine (DA) to oxidized DA (oxDA, color in orange) in the presence of hydrogen peroxide, which can obtain a three-color colorimetric result due to the couple of complementary colors of blue and orange. The determination of Cu2+ is achieved based on the absorbance value at 460 nm of the composite peak of TMB+ and oxDA. After systematically optimizing the reaction conditions, the developed bisubstrate colorimetric method exhibits high sensitivity for the detection of Cu2+ (limit of quantitation (LOQ) is 0.0625 μM) with a wide linear range of 0.0625–8.0 μM (R2 = 0.991). Meanwhile, the reaction solution shows an orange, brown, and green color change with the increase in Cu2+ concentration, which can realize the semi-quantitatively detection by the naked eyes. Furthermore, TMB was also used as a single substrate for Cu2+ detection with a linear range of 0.5–9.0 μM (R2 = 0.994), and the LOQ is determined to be 0.5 μM. The LOQ of bisubstrate system is 8 times lower than that of single substrate system, indicating a higher sensitivity for the detection of Cu2+ using the bisubstrate colorimetric system. In addition, the developed bisubstrate colorimetric method exhibits excellent selectivity for Cu2+ detection, which was applied in the determination of Cu2+ in lake water and soil samples, and satisfactory spiked recoveries were obtained.