Microcalorimetric methods for substrate determination in flow systems with immobilized enzymes

Area of Science:

• Biochemistry and Analytical Chemistry
• Enzyme Catalysis and Calorimetry

Background:

• Accurate quantification of biological analytes like urea and glucose is crucial in various scientific fields.
• Traditional analytical methods may require complex sample preparation or lack sensitivity for low concentrations.

Purpose of the Study:

• To develop a sensitive calorimetric method for determining urea and glucose concentrations.
• To explore the use of immobilized enzymes in a flow calorimeter for enhanced analytical performance.
• To demonstrate the principle of coupling enzyme reactions to increase sensitivity.

Main Methods:

• Utilized a LKB-flow calorimeter with immobilized enzymes in a reaction cell.
• Employed an "enzyme thermistor" system featuring a flow-through cell with immobilized enzyme and thermistors.
• Measured the enthalpy of enzyme-catalyzed reactions to quantify analytes.

Main Results:

• Successfully determined urea in the range of 0.5-5 mumol and glucose from 0.03-0.5 mumol with 2-5% accuracy.
• Achieved analysis times of 20 minutes per sample.
• Demonstrated glucose determination in the range of 0.05-1 mumol using the enzyme thermistor based on temperature differences from reaction heat.

Conclusions:

• The enzyme thermistor provides a sensitive and accurate method for determining urea and glucose.
• Calorimetric measurements using immobilized enzymes are effective for quantitative analysis.
• Coupling enzyme reactions can significantly improve sensitivity in calorimetric determinations.