Bst buffer

Optimization of the LAMP assay was performed in a 25 µL reaction mixture containing 2.5 µL 10× Bst buffer (New England Biolabs, Ipswich, MA, USA), 0.2 pmol each of F3 and B3, 0.8 pmol each of FIP and BIP, different concentrations of dNTPs (0.4, 0.6, 0.8, 1, and 1.2 mM), different concentrations of MgSO₄ (3, 4, 5 and 6 mM), 8 U Bst DNA polymerase (New England Biolabs), and different concentrations of DNA template. The reaction mixture was incubated at 60°C–65°C for different times and heated to 95°C for 5 minutes to stop Bst DNA-polymerase activity in the reaction. L. pneumophila serogroup 1 (ATCC 33152) was used as the positive control. Finally, LAMP-reaction products were electrophoresed on 2% agarose gel for detection of best LAMP product. After optimization of the LAMP assay, the amplified products were detected by adding 1 µL 1:10 SYBR green I (Thermo Fisher Scientific) into a reaction microtube. The reaction was considered positive if its color turned from orange to green under ultraviolet light.16 (link)

N-isopropylacrylamide (NIPAM; Wako Pure Chemical Industries Ltd.) was recrystallized from n-hexane before use. For hydrogel synthesis, N,N′-methylene-bis-acrylamide (MBA), ammonium peroxydisulfate, and N,N,N′,N′-tetramethyl ethylenediamine (TEMED) were used as a cross-linker, initiator, and accelerator, respectively, and they were purchased from Sigma-Aldrich and used as received. PSA polymer with a molecular weight of 800 kDa was obtained from Nippon Shokubai Pte Ltd. and used as received. Branched PEI (Sigma-Aldrich) with a molecular weight of 2 and 25 kDa, TMC (Sigma-Aldrich), and hexane were used for the synthesis of the PA layer on the hydrogel surface. Droplet production oil and stabilizer were obtained from Suzhou Rainsure Scientific Co. Ltd. Adenosine 5′-phosphosulfate, adenosine 5′-triphosphate sulfurylase, Bacillus stearothermophilus (Bst) polymerase v2.0 Warm Start, and 10× Bst buffer were purchased from New England Biolabs (NEB), Beijing, China. d-Luciferin sodium was obtained from Promega (Shanghai, China). Avian myeloblastosis virus Reverse Transcriptase was purchased from Woosen Biotechnology (Shanghai, China).