Nanoporous alumina membranes

Nanoporous alumina membranes were purchased from Whatman, Inc., Maidstone, UK. The membranes were round with 13 mm diameter and were 60 μm thick. The nanopores were about 100 nm in diameter. Histamine and anti-Histamine antibody produced in rabbits were purchased from Sigma Aldrich (St. Louis, MO, USA). (3-glycidoxypropyl) trimethoxysilane (GPMS, 98%), N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), trichloroacetic acid (99%), sodium hydroxide (98%), and n-butanol (99.8%) were also bought from Sigma Aldrich. Dimercaptosuccinic acid (DMSA) modified Fe₃O₄ MNPs were obtained from XFNANO (Nanjing, China). Toluene (99.8%), anhydrous ethanol (99%), and hydrogen peroxide (30%) were purchased from Sigma Aldrich. All of these chemicals were used as received without further purification.

Nanoporous alumina membranes with 200 nm nanopore diameter and 60 µm thickness (Whatman, Inc., Maidstone, UK) were functionalized to detect bacterial response to antibiotics. Nanoporous membranes were modified by GPMS (98%, Sigma-Aldrich, Darmstadt, Germany) for antibody conjugation according to the procedures described in the previous study [26 (link)]. In brief, membranes were boiled in hydrogen peroxide (H₂O₂, 30%, Sigma-Aldrich) for 30 min. They were washed in DI water for 10 min and dried. Then, they were silanized by GPMS in the mixture of toluene and GPMS (2%) at 60 °C overnight. The membranes were rinsed by toluene (99.8%, Sigma-Aldrich) and followed by anhydrous ethanol (99.8%). It repeated for three times. The membranes were cured for 2 h at 60 °C and the functional epoxy groups formed on the membrane surfaces.

Specifically, 8-OHdG, bovine serum albumin (BSA), citric acid, diethylenetriamine (EDTA), (3-glycidyloxypropyl)trimethoxysilane (GPMS), glutaraldehyde, methylbenzene, chloroauric acid, 3-mercaptopropionic acid (MPA), zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O), methanol (99.8%), sodium chloride, potassium chloride, calcium chloride, acetone, N,N-dimethylformamide (DMF), sodium borohydride (NaBH₄, 99.99%), and dehydrated alcohol were obtained from Sigma Aldrich (St. Louis, Missouri (Mo), USA). NaCl, KCl, CaCl₂, MgCl₂, thymine, cytosine, adenine, guanine, and hydrogen peroxide were ordered from ALADDIN Reagent (Shanghai, China). Nanoporous alumina membranes were purchased from Whatman (Boston, Massachusetts (Ma), USA). Alongside, 8-OHdG antibody was bought from Abcam (Cambridge, UK). In addition, 2-methylimidazole (2-MeIM, 99%), 1-dodecanethiol (DDT, ≥98%), hexadecyltrimethyl ammonium bromide (CTAB, 98%), silver nitrate (AgNO₃, ≥99.0%), 11-mercaptoundecanoic acid (MUA, 95%), and 4-nitrophenol (4-NP, ≥99.5%) were purchased from Sigma Aldrich (St. Louis, Missouri (Mo), USA). The chemicals were used as received without further purification.