Electroanalysis of Enzyme Activity in Small Biological Samples: Alkaline Phosphatase
ANALYTICAL CHEMISTRY(2021)
摘要
The discovery of sulfite-stabilized anodic current of hydroquinone (HQ) at high pH was used to develop two new methods for measuring the activity of the key biomarker alkaline phosphatase (ALP). Both approaches relied on the monitoring of ALP-triggered release of HQ from a substrate hydroquinone diphosphate (HQDP) into a pH 10.00 solution. One detected the released HQ via the internally calibrated electrochemical continuous enzyme assay (ICECEA) at a glassy carbon (GC) electrode with no sample incubation. The other used sample incubation with HQDP and quantified the released HQ via a coulometric assay at a commercial glucose test strip (GTS). The assay solution was optimized by investigating the ALP/HQDP/HQ system at a GC electrode. The ICECEA revealed high affinity of ALP for HQDP (K-m(app), 87 mu M; V-max, 0.36 mu M min(-1)) and detected ALP down to 0.022 U L-1. At GTS, ALP was detected down to 0.064 U L-1 in a 1 mu L sample of human serum after a 20 min incubation at room temperature. The linear range (R-2, 0.994) extended at least up to 1.7 U L-1 ALP, which covered more than the clinical range for ALP in serum. The interferences from the sample matrix including those from indigenous glucose were eliminated using a charge difference Delta Q (=Q(total) - Q(sample matrix)) as a signal for ALP. Both advances proposed here are direct (no auxiliary enzymes or labels required), accurate (98 +/- 3% ALP signal recovery), and precise (relative standard deviation (RSD), <7%). The HQDP-GTS-based assay advances the analysis of ALP activity in microsized real-life samples.
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关键词
Alkaline Phosphatase,phosphate,hyperphosphatemia,Hypophosphatasia
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