Toward the point-of-care testing of alkaline phosphatase in human serum

This work addresses challenges of simple but reliable determination of enzyme alkaline phosphatase (ALP) in μL-sized serum samples. The single-tube assay and plate-based immunoassay were developed by using a commercial glucose test strip (GTS) to transduce analytical signal from the ALP + glucose-6-phosphate (G6P) reaction. The glucose released from G6P contributed to the flow of anodic charge Q_20s through GTS. The interferences from serum matrix including those from native glucose were eliminated by devising a signal ΔQ_20s = Q_{20s (total)} - Q_{20s (matrix)}. The ΔQ_20s was proportional to ALP activity within a linear range of 7.4-720 IU L^-1 for assay (R², 0.995) and 10-300 IU L^-1 for immunoassay (R², 0.983), which far exceeded a normal clinical range for ALP in adult human serum (30-150 IU L^-1). Both methods required no auxiliary enzymes or labels, and were accurate (93-99 % signal recovery) and precise (RSD ≤10%). The assay was a simpler option because it required only one 20-min incubation step to determine ALP. The immunoassay involved three steps but it was still less laborious (by ∼70%) than a traditional enzyme-linked immunosorbent assay (ELISA) of ALP due to the substitution of ELISA detection antibody with GTS. The GTS-G6P-based methods bring ALP testing closer to the point-of-care locations, which is important considering the growing role of ALP as a prophylactic, therapeutic, and anti-inflammatory agent, and a marker of human diseases.