Manganese chloride

The genomic DNAs (gDNAs) of bacteria used in the reference assays were extracted by chemical methods using a TIANamp Bacteria DNA Kit (Tiangen Biotech, Beijing, China). The concentrations of the purified gDNAs were calculated using a Nanodrop1000 spectrophotometer (ThermoFisher Scientific, USA) and diluted with water to desired concentrations. The 2 × LAMP mastermix was provided by CapitalBio Corporation (Beijing, China), and bovine serum albumin (BSA, final concentration of 3 mg mL⁻¹) was added to the mastermix (total volume of 60 μL) to decrease the non-specific adsorption of enzymes^{40 (link)}. Calcein, manganese chloride, and BSA were purchased from Sigma-Aldrich (Shanghai, China). The DL 2000 DNA marker was purchased from TaKaRa (Dalian, China), and GeneGreen dye was purchased from Tiangen Biotech (Beijing, China). The gel and chip images were obtained and processed using a gel imager (C150, Azure Biosystems, USA). All LAMP primer pairs were synthesized by Invitrogen (Beijing, China), and the sequences are listed in Supplementary Table S1. Each LAMP reaction required four to six primers named F3, B3, FIP, BIP, LF, and LB (LF and LB are not necessary), and the final concentration of each primer in our experiments was 0.3, 0.3, 2.4, 2.4, 1, and 1 μM, respectively.

Bordetella pertussis Tohama I strain (59 (link)) and its derivatives (see Table S1 in the supplemental material) were grown on Bordet-Gengou agar (BGA) plates supplemented with 15% sheep blood for 3 days at 37°C prior to inoculation into liquid medium. For liquid cultures, bacteria were grown in Stainer-Scholte (SS) medium (42 (link)) supplemented with 0.1% cyclodextrin (Sigma-Aldrich) and 0.5% Casamino Acids (Difco) in an Innova 43 orbital incubator (Eppendorf) at 37°C and 160 rpm. When required, SS medium was supplemented with various concentrations of manganese chloride (Sigma-Aldrich) as specified in figure legends. Strains bearing the pBBRMCS1 vector were grown on BGA plates supplemented with chloramphenicol (10 μg ml⁻¹; Sigma-Aldrich).

Tannic acid (TA) and copper nitrate trihydrate were purchased from Marcklin Biochemical Co., Ltd. Pluronic F127 was purchased from Sigma-Aldrich. The Pb²⁺ and Epn molecule binding DNA probe was synthesized using integrated DNA technology. The following DNA sequence was used: 5′-CCTGGGCGGGTAGGGCGGGATCGGGTCCAGGT. A stock solution of DNA was prepared via dissolving 50 mM DNA in Tris hydrochloride (Tris–HCl) buffer solution (pH = 7.5) and it was stored at 4 °C. Epirubicin, lead nitrate, mercury nitrate, calcium chloride, nickel chloride, cobalt chloride, copper chloride, barium chloride, iron chloride, magnesium chloride, manganese chloride, imatinib (IMT), ampicillin (AMP), streptomycin (STR), tamoxifen (TMF), and other standard chemicals were purchased from Sigma-Aldrich (India) and SRL Pvt Ltd (India). The stock buffer and metal salt solutions used in the experiments were prepared using double-distilled water.

Next, 100-μL portions of Fusarium sp. DS 682 spore suspension were added to M9 PDA plates, followed by incubation at 28°C for 2 weeks. M9 PDA medium was prepared by adding micronutrients (see recipe below), 24 g/L potato dextrose (BD Biosciences, San Jose, CA), and 2% granulated agar (BD Biosciences) to M9 salts (MP Biomedicals, Irvine, CA) in Millipore water. The micronutrients consisted of 0.145 mM ammonium molybdate (Thermo Fisher Scientific), 2 mM boric acid (Sigma-Aldrich, St. Louis, MO), 0.151 mM cobalt chloride (Sigma-Aldrich), 0.048 mM cupric sulfate (Sigma-Aldrich), 0.404 mM manganese chloride (Sigma-Aldrich), and 0.048 mM zinc sulfate (Thermo Fisher Scientific). These micronutrients were added after autoclaving the M9 PDA solution, when the solution was ~55°C. After pouring the M9 PDA into petri dishes, the solidified agar plates were wrapped in Parafilm and stored at 4°C for future use. M9 PDA plates were primarily used to maintain the fungal cultures and not in the micromodel experiments described in this paper. The fungal experiments in the micromodel and for proteomics analysis were conducted using PDA plugs and PDA plates, respectively, as described in detail below.

Reagents (of analytical purity) Chelex-100 and betaine were purchased from Sigma, USA; manganese chloride, magnesium sulfate, potassium chloride, sodium hydroxide, EDTA, and ammonium sulfate from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); Tris-HCl from the Shanghai Shengke Biotechnology Co., Ltd. (Shanghai, China); Triton X-100 from the Beijing Meilaibo (MyLab) Medical Technology Co., Ltd. (Beijing, China); dNTPs from Pharmacia, USA; NP-40 from Fluka, USA; 2× Tap MIX Kit from the Tiangen Biochemical Technology Co., Ltd. (Beijing, China); Agarose from Amresco, USA; LAMP DNA Amplification Kit, LAMP reaction tube, and calcein (the fluorescent detection reagent) from the Eiken Chemical Co., Ltd. (Tochigi, Japan); the real-time turbidity meter (LA-320C) from the Rongyan Chemical Co., Ltd. (Tokyo, Japan); thermostatic metal bath (HB-2) from the Wealtech Corporation (Sparks, NV, USA); Spectrophotometer (NanoQ^TM) from the CapitalBio Technology Co., Ltd. (Beijing, China); gel imaging system (Quantun -ST5) from the Gel imager VILBER (Beijing, China); and PCR thermal cycler (ETC811) from the Gene Amplifier Suzhou Dongsheng Xingye Scientific Instrument Co., Ltd. (Suzhou, China).