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Optimization of an ammonia assay based on transmembrane pH-gradient polymersomes
Anastasia Spyrogianni1, Charlotte Gourmel1, Leopold Hofmann1
1Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 3, 8093, Zurich, Switzerland.
Scientific Reports
|November 12, 2021
Summary
This study developed a new microplate assay for measuring ammonia levels in patients with liver disease. The assay uses pH-gradient polymersomes and offers accurate quantification in biological fluids, aiding ammonemia monitoring.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Medical Diagnostics
Background:
- Accurate ammonia quantification is crucial for managing liver diseases and preventing hyperammonemia.
- Existing methods for ammonia measurement may lack precision or robustness in biological samples.
- Regulatory guidelines for bioanalytical method validation are essential for clinical assays.
Purpose of the Study:
- To develop and validate a microplate-based assay for precise and robust ammonia quantification in biological fluids.
- To ensure the assay meets regulatory standards for bioanalytical method validation.
- To address challenges in ammonia measurement, such as interference from bilirubin.
Main Methods:
- Development of a microplate assay utilizing transmembrane pH-gradient polymersomes encapsulating pH-sensitive ratiometric fluorophores.
- Utilizing four-parameter logistic regression for data analysis and establishing a wide quantification range.
- Testing assay selectivity against common interfering substances like amino acids and pyruvate.
- Adapting the assay by employing different fluorophores (pyranine and hemicyanine) to mitigate bilirubin interference.
Main Results:
- The developed assay demonstrated a broad quantification range of 30–800 μM ammonia.
- The assay showed high selectivity, with no interference observed from clinically relevant concentrations of amino acids or pyruvate.
- Successful quantification of ammonia in spiked human plasma samples was achieved, with results comparable to a commercial point-of-care device.
- The assay performance was optimized for samples with varying bilirubin levels by selecting appropriate fluorophores.
Conclusions:
- A novel, validated microplate assay for ammonia quantification in biological fluids has been successfully developed.
- The assay offers accuracy, precision, and robustness, making it suitable for monitoring ammonemia in liver disease patients.
- The adaptable design, particularly the use of different fluorophores, allows for reliable ammonia measurement even in the presence of bilirubin interference.
