Headspace paper-based analytical device for ammonia quantification in human biological samples

This article presents a simple and cost-effective headspace paper-based analytical device (hPAD) for the quantification of ammonia in human biological samples. The aim of this approach is to enhance the detection selectivity for ammonia in complex samples. The detection principle leverages basic chemistry, wherein ammonia reacts with copper sulfate (CuSO₄) to form the complex ion tetraamminecopper(II) sulfate ([Cu(NH₃)₄]SO₄), resulting in a colour change from pale blue to dark blue on a paper substrate. The quantitative analysis of ammonia is straightforward through placement of the sensor on the inside lid of the sample vial, and the resulting colour change is measured using a smartphone and image processing software. Upon optimization, the developed assay demonstrated a linear range between 2.5 and 40.0 μM (R² = 0.9955) with a detection limit (LOD) of 0.90 μM. The sensor also exhibited high precision, with the highest relative standard deviation (RSD) recorded at 6.17%. Moreover, the method showed remarkable selectivity, as the sensor showed no response to common interfering molecules in a complex biological matrix. The technique is fast, requiring only 4 min for the reaction, and does not necessitate any heating procedures. Furthermore, the developed method provides excellent accuracy for detecting ammonia levels in both human serum and urine samples, with recovery rates ranging from 93.4% to 107.6%. Therefore, the hPAD offers a simple and affordable solution by sensing in the headspace that overcomes the limitations of direct measurement in the sample, which may be affected by the colour, pH, other existing ions and molecules in the sample solution. Overall, this approach is suitable for various applications in both medical and environmental analysis.