Determining microbial extracellular alkaline phosphatase activity in seawater based on surface-enhanced Raman...

Microbial extracellular alkaline phosphatase (ALP) plays a significant role in marine phosphorus cycle. Therefore, it is of paramount importance to accurately and rapidly measure ALP activity (APA) in seawater. However, the applications of the existing APA measurement methods are constrained by cumbersome pre-processing, lengthy measurement times, and the influence of colored substances or suspended particles in seawater samples, which limit our accurate understanding of the marine phosphorus cycle. In this study, we developed a sensitive and rapid technique for the quantitative determination of microbial alkaline phosphatase activity in seawater based on surface-enhanced Raman spectroscopy (SERS). This method uses 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as the substrate, and dimethyl sulfoxide (DMSO) as an internal standard to establish a model for quantifying APA in seawater samples. Our results show that the Raman intensity ratio (I₆₀₀/I₇₀₀) between the enzymatic reaction product 5-bromo-4-chloro-3-indole (BCI oxide dimers) (I₆₀₀) and the internal standard (I₇₀₀) is an ideal quantitation parameter, and there is a strong linear relationship between I₆₀₀/I₇₀₀ (y) and APA (x): y = 0.301x + 1.105, R² = 0.981. This method is capable of determining APA over a dynamic range of five orders of magnitude (from 0.1 to 10⁴ mU L^-1) with a detection limit of 0.1 mU L^-1. The reliability of the method is confirmed by comparing the kinetic parameters of the fluorogenic method. Further, this method was tested and successfully applied to quantify APA in coastal and open ocean seawater samples from the Western Pacific Ocean, demonstrating the potential of this method for rapid and reliable detection of APA in the marine environment.