Our proposed end users are the general public with the objective to narrow the distance between science and society, thus, our device has to be user friendly, non-expensive, portable, among others. However, it is also meant to be useful in scientific investigations if required. We envision our project as a portable device where someone can take a sample of any body water of interest and evaluate if there is a presence of the contaminants evaluated in a rapid way (erythromycin, pentachlorophenol, and rifampicin).

A microfluidic paper-based analytical device (μPAD) is a miniature laboratory analytical tool fabricated with paper material, and is capable of analyzing complex and small amounts of biochemical samples (Akyazi e tal., 2018). The advantages over other technologies are the fluid handling and analysis, low cost, ease of fabrication and operation, and equipment independence (Selvakumar & Kathiravan, 2021); advantages that align with the needs of our end users. The evaporation of the liquid sample when passing through the channel is generally considered a limitation feature of μPADs. Nonetheless, in this case, it could benefit sample pre-concentration when utilized in sample loading process, as the large specific surface area of paper fibers and the open channel of μPADs provide a perfect platform for air-liquid contact that could speed up evaporation easily (Jin et al., 2020).

Therefore, we proposed the implementation of a μPAD made of chromatographic paper or filter paper (Whatman Grade/No. 1, 3, 4, and 6) due to their moderately uniform thickness and superior pore size, with one sample deposit and the enzyme detector (Figure 1). The hydrophobic region will be generated using wax, and the enzyme detector will need to have a dried enzyme cocktail with a buffer in the optimal pH of our enzymes (erythromycin 12-hydroxylase and pentachlorophenol monooxygenase, optimal pH = 7.5, rifampicin monooxygenase optimal pH = 7, and diaphorase, optimal pH = 7.5) (Ilacas & Gomez, 2018). The enzymes can be immobilized onto the paper via pure adsorption, covalent conjugation, or polymeric entrapment. The efficacious enzyme immobilization on the paper-based platform is critical for the μPAD performance (Nadar et al., 2021). Then, we would have to evaluate the best performance between the paper and immobilization technique that accoplates to our colorimetric assay. In addition, to determine the amount of enzyme added in a paper-based assay we would have to consider the enzyme activity, as a small number of enzymes with a low turnover number would not provide a reliable output. Regarding the redox indicator, it should be in excess to avoid saturation of the signal by limited availability of reagent (Jin et al., 2020).

Based on the prototype proposed, the capillary activity of the paper together with the hydrophobicity of the wax, would direct our water sample into the enzyme detector. If the contaminants are present in the water, then the enzyme detector will turn blue as shown in Figure 1; if not, it will stay the same color. Finally, the results can be evaluated visually (if general public is using it) or using software, such as ImageJ (Aksorn & Teepoo, 2020).

In order to be safe, the samples to be used in the device should be taken with boots, pants, gloves, eye glasses, and a mask, as we would be treating with possibly highly contaminated water bodies.

The challenges we would need to consider is the possible loss of enzymatic activity when attached to the paper in comparison to its free form due to the unfavorable orientation of enzymes and accessibility issues offered by the carrier system, which confronts the sensitivity of enzyme-based analytic devices. Similarly, in most cases, the enzymes are not firmly anchored on the detection zone of mPADs, thus, we would need to genetically modified enzymes by incorporating appropriate binding domains, which adhere spontaneously to the paper (cellulose), or use biomineralization techniques (Nadar et al., 2021).

In addition, the optimization of parameters such as enzyme activity and stability, operational conditions, and the amount of sample for analysis and redox indicator should be experimentally tried to obtain accurate results in the device within a short duration (Nadar et al., 2021). Finally, it is important to consider the incompatibilities that could arise when coupling two biological assays, such as the optimal conditions at which each enzyme operates, as the optimum condition for one system may be not ideal for another (Gianini-Morbioli et al., 2017).