Quantification of alkaline phosphatase in cell lysates by a simple fluorescence method in combination with a modified standard addition calibration strategy

Abstract

Alkaline phosphatase (ALP) is an important diagnostic indicator of many human diseases. Its detection is of great importance in fields such as clinical diagnosis and biomedical research. In this contribution, a simple label-free fluorescence sensing method for ALP detection was designed by integrating ALP triggered hydrolysis, terminal deoxynucleotidyl transferase assisted DNA polymerization and complex formation between G-quadruplex and thioflavin T together. Furthermore, in order to mitigate the influence of matrix effects and background interference effects on the quantitative results of the proposed fluorescence sensing method encountered in the analysis of real-world complex biological samples, the probe technique-based generalized multivariate standard addition strategy (GMSAprobe) was specifically modified, and combined with the proposed fluorescence sensing method to achieve satisfactory quantitative results for ALP in cell lysate samples with accuracy comparable to that of a commercial ALP assay kit. Its limit of detection and limit of quantification values were about 0.02 and 0.07 U/L, respectively, more than 20 times lower than those of the commercial ALP assay kit. Due to its merits of simplicity, sensitivity, specificity, as well as robustness to matrix effects and background interference effects, the combination of the proposed fluorescence sensing method and the modified GMSAprobe has a great potential for ALP assay in biochemical research and clinical diagnosis.