Smartphone-adapted multianalyte biosensor platform for fluorescent analysis of human biomarkers and immunosuppressive drugs using PQQ- and NAD⁺-dependent enzymes

Here we describe a multianalyte biosensor platform for fluorescent analysis of different human state biomarkers (α-amylase, phenylalanine, glucose, lactate/pyruvate, alcohol) and some immunosuppressive drugs (cyclosporine A, tacrolimus, methotrexate, rapamycin) using chimeric PQQ- and natural NAD⁺-dependent enzymes. The principle of the approach is based on the analysis of the brightness of photography of the sensor plate taken with a smartphone camera and processed via ImageJ software. The brightness of the image correlates with the fluorescence intensity of the sensor’s spots which is produced by enzymatic reduction of phenazine methosulfate or its derivative used as a fluorescence probe at UV 356 nm irradiation, where the amount of the reduced dye depends on the concentration of the target analyte (the enzymatic substrate) in the tested sample. The sensor plate is composed of simple and cheap components, while the procedure of its preparation and usage is easy and does not require any specific skills or expensive instrumentation. The proposed sensor platform is characterized by a high selectivity and storage stability depending on the selectivity and stability characteristics of the used enzyme in an immobilized state. The proposed sensor platform could be used for precision quantitative analysis of a single (or several) analytes or used for simultaneous qualitative multianalyte assay of numerous of them via Boolean Logic Gates.