Microfluidic device proposal

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• Reheat the printed wax on a hot plate of 120°C for 5 minutes to create the hydrophobic barriers as the wax covers the total thickness of the paper  (Kaarj, et al., 2018).
• Remove the paper from the hot plate and allow it to cool at room temperature (Carrilho, et al., 2009).
• Mix 250 µl of the sample with 500 µl a solution of 0.2N NaOH and 1% SDS (Feliciello, et al., 1993)
• Load 50 µl of the supernatant onto the loading chamber and wait for it to travel through the channel and reach the reaction chamber. Large components (debris) will remain on the loading chamber and channel, and DNA will reach the reaction chamber based on the capillary forces, paper porosity, and strong negative charges. (Kaarj, et al., 2018).
• Cut the paper strip to isolate the reaction chamber, without touching the inside of the chamber.
• Load 15 µl of the LAMP MasterMix (1.6 µmol/L of each of the inner primers FIP and BIP, 0.2 µmol/L of each of the outer primers F3 and B3, and 0.8 µmol/L of each of the loop primers LF and LB. 1.2 mmol/L each dNTP, 6 mmol/L MgSO4, 1 × Bst DNA polymerase buffer) onto the reaction chamber (Wang, et al., 2015; Kaarj, et al., 2018).

For CTX-M-15:

1. LF: CCGATGTACCCAGCGTCAGATTCCGACATCGG (Figure 4)
2. LB: ATCGTAGCATTCAGGCTGGACTGCCTACGAT (Figure 5)
3. F3: GTGATACCACTTCACCTCGG
4. B3: GTTGGTGGTGCCATAGCC
5. FIP (F1c+F2): ATCCATGTCACCAGCTGCGCGCAATGGCGCAAACTCTG
6. BIP (B1c+B2):GAAAGGCAATACCACCGGTGCATATCCCCCACAACCCAGG

TaqMan molecular beacons (hairpin structure) were employed to work as loop primers (LF and LB) labeled with 6-FAM on the 5’ end and a Black Hole Quencher (BHQ) on the 3’ end. The hairpin structure allows proximity between the 6-FAM fluorophore and the BHQ, impeding fluorescence emission. Once the primer has annealed to the target structure, proximity is lost and green fluorescence is emitted. However, once the primer has been displaced by the exonuclease activity of the polymerase, the increased physical separation between the 6-FAM and the BHQ allows enhanced fluorescence emission detected by UV light (Varona and Anderson, 2019; Zhang, et al., 2023).

•  Seal the strip with glass and parafilm to avoid evaporation  (Kaarj, et al., 2018).
• Position the devices (experiment and negative control) covered on top with a thin styrofoam    layer on a hot plate at 65°C for 35 mins to achieve amplification (Figure 6)  (Byers, et al., 2020; Kaarj, et al., 2018).