Fluorescent Biosensing Of Alkaline Phosphatase Based On Nucleic Acid Probe

Objective:Biosensing provides a novel scheme for early diagnostics and biomarker detection,in which nucleic acid probes can serve as feasible tools due to their excellent chemical properties,modifiability,and lower cost.Alkaline phosphatase（ALP）is an important biochemical marker that is of great significance in the clinical laboratory and disease diagnosis.At present,with the in-depth research,there is an emerging requirement for the development of enzyme activity biosensing platforms with higher sensitivity and efficiency.Therefore,two nucleic acid probe-based fluorescent biosensors were developed in this paper to quantify ALP activity,combining nanomaterials and signal amplification techniques,respectively.And the detection performance was evaluated.Methods:（1）A MnO₂nanosheets mediated G-quadruplex/thioflavin T fluorescence method was constructed.The dephosphorylation ability of ALP was used to generate ascorbic acid,which can reduce MnO₂nanosheets to Mn²⁺,thus releasing the previously-absorbed nucleic acid probe and forming G-quadruplex/thioflavin T structures in the system,then the fluorescent signals were produced.（2）Construction of a sensitive fluorescence method based on terminal deoxynucleotidyl transferase（Td T）mediated-template free polymerization and T7exonuclease-assisted target recycling amplification.ALP could specifically hydrolyze the 3’-phosphate modification of the DNA substrate,achieving signal conversion and initial amplification by Td T extension.After the addition of Taqman probe and T7 exonuclease,the second-time signal amplification was developed.Fluorescence was generated from the cleavage of Taqman.Results:Both the two methods were able to quantify the concentration of ALP,with a linear range of 0-5 and 0.01-8 U/L,LOD of0.0399 and 0.0074 U/L,respectively.Experiments of selectivity,inhibition,and recovery showed satisfying results.Real serum samples detection was completed,and the results were consistent with those obtained from the clinically-used method.Conclusion:In this paper,two fluorescent biosensors for ALP based on nucleic acid probes were successfully constructed,with excellent sensitivity,high efficiency,and lower cost.These methods have shown the potential applicability for inhibitors screening and biological samples detection,providing new perspectives for point-of-care testing,disease diagnosis,and drug discovery.