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Research Article
Jean-Christophe Leroux
ETH Zurich
Corresponding Author
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Reliable ammonia quantification assays are essential for monitoring ammonemia in patients with liver diseases. In this study, we describe the development process of a microplate-based assay for accurate, precise, and robust ammonia quantification in biological fluids, following regulatory guidelines on bioanalytical method validation. The assay is based on transmembrane pH-gradient polymersomes that encapsulate a pH-sensitive ratiometric fluorophore, the fluorescence signal of which correlates with the ammonia concentration in the sample. Using four-parameter logistic regression, the assay had a large quantification range (30–800 µM ammonia). As for selectivity, the presence of amino acids or pyruvate (up to clinically relevant concentrations) showed no assay interference. In samples with low bilirubin levels, polymersomes containing the fluorophore pyranine provided accurate ammonia quantification. In samples with high bilirubin concentrations, billirubin’s optical interference was alleviated when replacing pyranine with a close to near-infrared hemicyanine fluorophore. Finally, the assay could correctly retrieve the ammonia concentration in ammonia-spiked human plasma samples, which was confirmed by comparing our measurements with the data obtained using a commercially available point-of-care device for ammonia.
Keywords
ammonia, Transmembrane , Polymersomes, microplate-based assay
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The full text of this article is available to read as a PDF.
Competing interest reported. JCL is a cofounder and shareholder of Versantis AG. LH is now an employee of Versantis AG. All other coauthors do not have any conflict of interest.
This is a list of supplementary files associated with this preprint. Click to download.
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See the published version of this preprint at Scientific Reports.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
Jean-Christophe Leroux
ETH Zurich
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Scientific Reports.
Version 1
Posted 26 May, 2021
No community comments so far
Editorial decision: Major revision
On 28 Sep, 2021
Reviews received
Received 25 Sep, 2021
Reviews received
Received 17 Jul, 2021
Reviewers agreed
On 04 Jul, 2021
Reviewers agreed
On 04 Jul, 2021
Reviewers invited
Invitations sent on 04 Jul, 2021
Editor assigned
On 28 Jun, 2021
Editor invited
On 20 May, 2021
Submission checks complete
On 20 May, 2021
First submitted
On 19 May, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Scientific Reports.
Reliable ammonia quantification assays are essential for monitoring ammonemia in patients with liver diseases. In this study, we describe the development process of a microplate-based assay for accurate, precise, and robust ammonia quantification in biological fluids, following regulatory guidelines on bioanalytical method validation. The assay is based on transmembrane pH-gradient polymersomes that encapsulate a pH-sensitive ratiometric fluorophore, the fluorescence signal of which correlates with the ammonia concentration in the sample. Using four-parameter logistic regression, the assay had a large quantification range (30–800 µM ammonia). As for selectivity, the presence of amino acids or pyruvate (up to clinically relevant concentrations) showed no assay interference. In samples with low bilirubin levels, polymersomes containing the fluorophore pyranine provided accurate ammonia quantification. In samples with high bilirubin concentrations, billirubin’s optical interference was alleviated when replacing pyranine with a close to near-infrared hemicyanine fluorophore. Finally, the assay could correctly retrieve the ammonia concentration in ammonia-spiked human plasma samples, which was confirmed by comparing our measurements with the data obtained using a commercially available point-of-care device for ammonia.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
The full text of this article is available to read as a PDF.
Competing interest reported. JCL is a cofounder and shareholder of Versantis AG. LH is now an employee of Versantis AG. All other coauthors do not have any conflict of interest.
This is a list of supplementary files associated with this preprint. Click to download.
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