Development of a Polymersome Blood Ammonia Assay Coupled with a Portable Near-Infrared Fluorometer

Abstract

Ammonia is a key biomarker in inborn and acquired liver disease. As clinical point-of-care blood ammonia assays are lacking, we developed a polymersome formulation for point-of-care blood ammonia sensing combined with a portable fluorometer. A pH-sensitive near-infrared (NIR) fluorescent dye was identified, which showed a strong fluorescence increase at acidic pH values. Building on reports on ammonia-selective PS-b-PEG polymersomes, these polymersomes were loaded with the NIR dye. These NIR fluorescent polymersomes sensed ammonia in a clinically relevant range in ammonia-spiked fresh whole blood with high linearity (R ² = 0.9948) after 5 min using a conventional tabletop plate reader. Subsequently, the assay was tested with a portable fluorometer. An ammonia-dependent fluorescence increase was detected in ammonia-spiked fresh mouse blood after 5 min using the portable fluorometer. The NIR dye-loaded PS-b-PEG polymersomes rapidly sensed ammonia with high linearity in whole blood. This assay was successfully combined with a portable fluorometer and only required 3 μL of blood. These findings motivate a further development and clinical translation of this point-of-care blood ammonia assay.

Figure 1

Polymersome ammonia assay and portable fluorometer. Mechanism of action of polymersome ammonia assay: ammonia diffuses across the membrane of transmembrane pH-gradient poly(styrene)-b-poly(ethylene glycol) polymersomes into the acidic core. The protonation of ammonia increases the luminal pH and the fluorescence intensity of an encapsulated pH-sensitive NIR fluorescent dye (A). In the proposed ammonia assay setup, capillary blood is collected by a fingerstick and added to a polymersome assay solution. The NIR fluorescence change is detected by a portable fluorometer (B). Portable fluorometer used in this study compared with an iPhone 13 as a size reference (C). NIR: near-infrared fluorescent.

Figure 2

pH-dependent fluorescence of IRDye 680RD. Fluorescence intensity of IRDye 680RD at different pH values detected using a tabletop plate reader (A) and a portable fluorometer (B). IRDye 680RD concentration: 2 μM. All results are mean ± SD (n = 3).

Figure 3

Ammonia sensing in ammonia-spiked buffer and mouse blood was carried out using a plate reader. Fluorescence intensity of IRDye 680RD-containing PS-b-PEG polymersomes at different volume fractions in ammonia-free isotonic phosphate buffer at pH 7.4 (A). Fluorescence intensity ratio of IRDye 680RD-containing PS-b-PEG polymersomes in ammonia-spiked buffer or blood over time (B). Fluorescence intensity ratio: fluorescence intensity in 0.5 mM ammonia-spiked buffer or blood normalized to nonspiked buffer or blood. Inner phase composition: dye concentration: 19 μM; buffer composition of inner phase: isotonic citric acid buffer 2.5 mM at pH 2.0; whole blood volume fraction (B): 3% (v/v); incubation at room temperature. Detector: tabletop plate reader. All results are shown as means ± SD (n = 3–4).

Figure 4

Ammonia sensing in ammonia-spiked mouse blood using a plate reader. Fluorescence intensity ratio of IRDye 680RD-containing PS-b-PEG polymersomes in ammonia-spiked blood over time (A). Fluorescence intensity ratio of IRDye 680RD-containing PS-b-PEG polymersomes in ammonia-spiked blood at 5 min with regression curve (B; data extracted from A). Fluorescence intensity ratio: fluorescence intensity of ammonia-spiked blood normalized to nonspiked blood. Inner phase composition: dye concentration: 37 μM; buffer composition of inner phase: isotonic citric acid buffer 2.5 mM at pH 2.0; whole blood volume fraction: 3% (v/v); incubation at room temperature. Detector: tabletop plate reader. All results are means ± SD (n = 4).

Figure 5

Ammonia sensing in ammonia-spiked mouse blood using a portable fluorometer. Fluorescence intensity ratio of IRDye 680RD-containing PS-b-PEG polymersomes in ammonia-spiked isotonic phosphate buffer of 50 mM at pH 7.4 over time (A). Fluorescence intensity ratio of IRDye 680RD-containing PS-b-PEG polymersomes in ammonia-spiked blood over time (B). Fluorescence intensity ratio: fluorescence intensity in ammonia-spiked buffer or blood normalized to nonspiked buffer or blood. Inner phase composition: dye concentration: 37 μM; buffer composition of inner phase: isotonic citric acid buffer 2.5 mM at pH 2.0; whole blood volume fraction (B): 3% (v/v); incubation at room temperature. Detector: portable fluorometer. All results as means ± SD (n = 4). **p < 0.01, ***p < 0.001.