Selective measurement of valine, leucine, and isoleucine using corresponding aminoacyl-tRNA synthetases and application to paper-based analytical devices for colorimetric detection

Background

Branched-chain amino acids (BCAAs) are vital for maintaining muscle performance and their measurement is critical for assessing nutritional status, health, and disease conditions. However, selective analysis of individual BCAAs remains challenging due to the limited selectivity of conventional enzymes. In this study, we explore the enzymatic detection of BCAAs (valine (Val), leucine (Leu), and isoleucine (Ile)) among 20 amino acids using their corresponding aminoacyl-tRNA synthetases (aaRSs) as recognition elements. Based on our findings, a paper-based analytical device (PAD) for Val, Leu, and Ile was developed using filtration paper.

Results

The calibration curves, and selectivity of each aaRS were assessed using a microplate reader. Trinder's reagent was used as the colorimetric reaction following aaRS reaction. Additionally, Val, Leu, and Ile analysis was conducted on real food samples to evaluate practical applicability. Linear relationships were established for Val (2–75 μM; correlation coefficient: R² = 0.959), Leu (2–50 μM; R² = 0.984), and Ile (1–100 μM; R² = 0.982), with detection limits of 18.5, 7.6, and 8.4 μM, respectively, using the microplate reader. Furthermore, PAD exhibited linear relationships for Val (7–80 μM; R² = 0.978), Leu (9–80 μM; R² = 0.956), and Ile (19–80 μM; R² = 0.961), with detection limits of 2.2, 3.1, and 6.3 μM, respectively. The analysis was completed within 15 min.

Significance

Trinder's reagent, a colorimetric reaction reagent, ensures ease of use and safety owing to its neutral pH. Furthermore, it facilitated the development of PAD and analysis of Val, Leu, and Ile. The findings of this study contribute to the advancement of robust biomedical devices precisely for amino acid analysis. High-resolution detection was accomplished using a USB camera and novel RGB value analysis equations.