Photometric and fluorometric alkaline phosphatase assays using the simplest enzyme substrates

Area of Science:

• Analytical Chemistry
• Biochemistry
• Bioanalytical Systems

Background:

• Alkaline phosphatase (ALP) activity is a key biomarker in various biological and clinical contexts.
• Existing methods for ALP determination can be costly and reagent-intensive.
• There is a need for affordable and efficient bioanalytical systems for routine ALP assays.

Purpose of the Study:

• To develop a highly cost-effective and fully-mechanized flow analysis system for alkaline phosphatase (ALP) activity determination.
• To utilize economic components like solenoid micropumps, microvalves, and dedicated optoelectronic detectors.
• To implement a simple and inexpensive detection scheme based on orthophosphate determination.

Main Methods:

• A flow analysis system was constructed using Arduino-controlled solenoid micropumps and microvalves.
• Dedicated optoelectronic flow-through detectors were employed for absorbance and fluorescence measurements.
• A molybdate method was used for orthophosphate determination, a product of ALP enzymatic conversion, utilizing simple and inexpensive reagents.
• Monofluorophosphate (MFP) was identified as a promising ALP substrate.

Main Results:

• The system achieved linear absorbance ranges of 100-600 U/L and fluorescence ranges of 30-100 U/L.
• Sensitivities were reported as 0.7 µV·L/U (photometry) and 2.3/1.0 µV·L/U (fluorometry).
• Limits of detection were as low as 5.1 U/L (photometry) and 0.9 U/L (fluorometry), with sample throughputs of 13-12 samples/h.
• ALP activity in serum samples showed good correlation with a reference method.

Conclusions:

• A highly cost-effective, mechanized flow analysis system for ALP activity determination has been successfully developed.
• The system's reliance on inexpensive components and a simple detection method makes it suitable for low-cost bioanalysis.
• The developed system demonstrates practical utility and accuracy for ALP assays in complex matrices like serum samples.