Development of a one-step analytical method for multiple amino acids using a microfluidic paper-based analytical device

In our previously reported LPAD for histidine, the enzymatic reaction had to be performed outside the LPAD: the reaction mixture with HisRS, histidine, ATP and MgCl2 in a microtube was heated using an aluminum heating block at 80 °C for 30 min and cooled on ice for 5 min. Then the reaction mixture was loaded onto the LPAD. The dummy step with heating the reaction mixture and pipetting the sample was necessary19. In this study, an LPAD that works consecutively for enzymatic and colorimetric reactions in one step.

Evaluation of filtration paper formats for microfluidics

The enzymatic reaction occurs when the enzymatic reaction mixture enters the detection zone; therefore, the enzymatic reaction time is important and will affect the LPAD response. The type and shape of filtration papers used for microfluidics were evaluated using Advantec Grade No. 1, Advantec Grade No. 5B, and MN616G. Table 1 shows the sets of lengths between the enzymatic reaction and detection zones and the width of the microfluidic paths. A length between the enzymatic reaction and the detection zones of 15 mm and the width of the microfluidic paths of 3.0 mm was determined to be the preferred size for the LPAD. A path of short length and/or narrow width showed no response or only a rare response when the reaction mixture reached the detection zone instantaneously. If the length was longer (20 mm), the loaded enzyme reaction mixture could not reach the detection area. Therefore, providing sufficient aaRS enzymatic reaction times when penetrating the reaction mixture into the filtration paper is important to consider when designing LPADs. The fiber density of the filtration paper is also an important factor. Advantec Grade No. 1 high density filter paper showed better performance at the point of fiber density as the reaction mixture penetrated gradually and this was sufficient for enzymatic reaction times. The high-density filter papers also retained the enzyme and reagent solutions sufficiently to allow the enzymatic reaction in the detection zone, indicating that the stable manufacture of LPADs might be possible. Further, at the time of the colorimetric detection which was performed based on the molybdenum blue reaction, the color depth changed in a time-dependent manner and became saturated. The evaluation of the color of the detection zones at 15 min after the deposition of the samples was preferred (data not shown).

Table 1 Evaluation of microfluidic and filtration paper sizes.

LPAD assay

The photos obtained after LPAD assay using 0 to 100 μM for each amino acid are presented in FIG. 3a. The color of the LPAD detection area for glycine (upper right corner of the LPAD) after loading glycine changed from yellow to blue, while the detection areas for tryptophan (upper left corner), histidine (lower left corner) and lysine (lower right corner) after glycine loading showed no color change. Similarly, the color of the detection zone of LPAD when only tryptophan, histidine or lysine were charged respectively, changed from yellow to blue and no reaction was observed for discordant amino acids.

picture 3

Photos of the laminated paper-based analytical devices (LPADs) after loading each amino acid. (a) Original image of each LPAD after interaction with 0–100 μM tryptophan, glycine, histidine and lysine. The color change was observed only in the substrate amino acid detection zone. (b) Images were color-inverted using the GNU image manipulation program.

Inverted images obtained using the GNU image manipulation program are shown in Figure 3b.

Figure 4 shows the standard curves for the detection of tryptophan, glycine, histidine and lysine (solid circle in each graph). The horizontal axis represents the initial concentration of each amino acid, and the vertical axis represents the integration signal (arbitrary unit), which is calculated as the product of brightness and detection area. The integration signal increased in response to the addition of substrate amino acids, and good linearity ranges between 3.6 and 100 μM were obtained for tryptophan, with a detection limit of 1.1 μM (r = 0.9717, Fig. 4a), 10.1–100 μM for glycine, with a detection limit of 3.3 μM (r = 0.9722, Fig. 4b), 5.9–100 μM for histidine, with a detection limit of 1.9 μM (r = 0.9816, Fig. 4c), and 5.6–100 μM for lysine, with a detection limit of 1.8 μM (r = 0.9756, Fig. 4d).

Figure 4
number 4

Calibration curves for the detection of tryptophan, glycine, histidine and lysine. The solid circle in each graph represents the substrate amino acid, while the open circles indicate the average of the integration signals of three non-substrate amino acids. Data represent the average of three measurements and error bars indicate standard deviations.

The limit of detection (LOD) of conventional HPLC (Hitachi Amino Acid Analyzer L-8900) is approximately 0.5 μM6, and slightly higher than the LOD of our LPAD. However, the measurable concentrations of each amino acid in the LPADs were within the approximate range of amino acid levels found in blood.

Figure 4 also shows the selectivity of LPAD. The open circles in each graph represent the average of the integration signal of three non-substrate amino acids; the open circle in Figure 4a (tryptophan detection area) indicates the average of the histidine, lysine and glycine integration signal. Each calibration curve was unslanted and the values ​​were almost the same as those of the 0 μM substrate amino acid; therefore, no response was observed for non-substrate amino acids. Due to the substrate specificities of TrpRS, GlyRS, HisRS and LysRS, these enzymes bind specifically to their corresponding substrate amino acids. Therefore, the LPAD could selectively analyze amino acids. In our previous article, no interference was observed in the binding of the substrate amino acid to aaRS. The binding activity of aaRS to the solo substrate amino acid and to the mixture of 20 amino acids was almost the same value; therefore, the existence of 19 other amino acids in the reaction mixture would not interfere with the binding of the substrate amino acid to aaRS14.

Validation of the LPAD

The reproducibility of LPAD responses to 100 μM of each amino acid among three different manufacturing (3 days) and dosing dates was assessed (Table 2). Each entry was repeated three times. The coefficient of variation [CV (%)] was approximately less than 2% and CV values ​​were low. These results suggest that the fabrication of LPADs, including the cutting of filtration papers and films, as well as the coating of reagents, can be reproduced with precision and consistency. The LPADs showed sufficient reproducibility for each amino acid. Additionally, as described above, they only required a few micromoles of each amino acid to function, which is consistent with blood amino acid levels.

Table 2 Reproducibility of Laminated Paper-Based Analytical Device (LPAD) responses to each amino acid among three different manufacturing and assay dates.
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