Electroanalysis of Enzyme Activity in Small Biological Samples: Alkaline Phosphatase

Area of Science:

• Biomarker detection
• Electrochemical biosensing
• Enzyme activity assays

Background:

• Alkaline phosphatase (ALP) is a key biomarker.
• Sulfite-stabilized anodic current of hydroquinone (HQ) at high pH was discovered.
• Existing ALP assays may require complex procedures or lack sensitivity.

Purpose of the Study:

• To develop novel, sensitive, and direct methods for measuring alkaline phosphatase (ALP) activity.
• To utilize the sulfite-stabilized anodic current of hydroquinone (HQ) for ALP detection.
• To create assays suitable for clinical diagnostics and microsized samples.

Main Methods:

• Developed two methods based on ALP-triggered release of HQ from hydroquinone diphosphate (HQDP).
• Method 1: Internally calibrated electrochemical continuous enzyme assay (ICECEA) using a glassy carbon (GC) electrode.
• Method 2: Coulometric assay using a commercial glucose test strip (GTS) after sample incubation.

Main Results:

• ICECEA detected ALP down to 0.022 U L⁻¹ with high affinity (Km app, 87 μM).
• GTS assay detected ALP down to 0.064 U L⁻¹ in human serum (1 μL) with a linear range up to 1.7 U L⁻¹.
• Both assays demonstrated accuracy (98 ± 3% recovery) and precision (RSD <7%), eliminating matrix interferences.

Conclusions:

• The developed methods provide direct, accurate, and precise quantification of ALP activity.
• The HQDP-GTS assay is particularly advantageous for analyzing microsized real-life samples.
• These advances offer improved tools for clinical diagnostics and biochemical analysis.