Dip-and-Read, Organic Solvent-Compatible, Paper-Based Analytical Devices Equipped with Chromatographic Separation for Indole Analysis in Shrimp

We developed an organic solvent-compatible paper-based analytical device (PAD) for the quantitative analysis of indole, which is an indicator of shrimp freshness. Although indole is insoluble in water, ethyl acetate is a suitable solvent to dissolve and extract indole from shrimp. The PADs are fabricated using a cutting method that allows the use of an organic solvent because no hydrophobic barrier is needed to form fluidic channels. Ehrlich's reagent consists of 4-(dimethylamino)benzaldehyde and p-dimethylaminobenzaldehyde and was deposited onto the reaction zone of the PAD followed by lamination to prevent evaporation of the ethyl acetate. Samples are introduced into the PAD via immersion in organic sample solutions. When the PAD is immersed into an indole solution of ethyl acetate in a closed bottle, the sample solution penetrates the channel of the PAD and successively flows into the detection zone to form a hydrophilic colored product. The PADs provide a linear relationship between the logarithm of the indole concentration and the color intensity within a range of 1.0-20 ppm with correlation coefficients of r²> 0.99. The limits of detection and quantification are 0.36 and 0.71 ppm, respectively. Relative standard deviations for both the intraday (n = 2) and interday (n = 3) precision were less than 2.5%. In the indole analysis of shrimp, the PADs separated the interfering orange-colored astaxanthin in the extract from the colored product of indole via the paper chromatographic principle. We used the PADs to investigate the degradation of shrimp, and the results showed a rapid increase in the indole level after 7 days. High-performance liquid chromatography verified the accuracy of the PADs by showing good agreement with the obtained indole levels.