Spectra por

The monomer (acrylamide, AAm), crosslinker (N,N-Methylenebis(acrylamide), MBA), and carbon nanospecies (carboxyl-functionalized multiwall carbon nanotubes, CNTs) were purchased from Sigma. Polyacrylamide (PAAm), with an average molecular weight of 3.76 × 10⁵ g/mol, was obtained in the laboratory through the free radical photopolymerization of the monomer in an aqueous solution in the presence of Irgacure 2959 (Sigma, St. Louis, MO, USA), purified through dialysis against distilled water (dH₂O) using dialysis membranes (SpectraPor, MWCO 12–14 kDa, Repligen, CA, USA). The polymer’s synthesis and the method employed for the assessment of its viscosity and average molecular weight are described in Appendix A. Ammonium persulphate (APS, Sigma) and trietanolamine (TEA, Sigma) were used as the redox initiating system of the AAm-MBA system. Phosphate-buffered saline (PBS 0.01 M, pH 7.4, Sigma-Aldrich, St. Louis, MO, USA) was prepared according to manufacturer’s instructions. For the antimicrobial tests, Staphylococcus aureus (ATCC 6538TM) and Escherichia coli (ATCC 10536TM) prepared according to the manufacturer’s recommendations were used as references. Casein soyabean digest broth (TSB, Sigma) was used as dispersant for the lyophilized bacteria, while casein soyabean digest agar (TSA, Sigma) was used as culture medium.

ADA was produced according to the method developed by Sarker et al. [17 (link)]. For this purpose, 5 g of sodium alginate (Art. No. 71238) was dissolved in 25 mL ethanol (99.8%), and 1.605 g sodium periodate was dissolved in 25 mL double distilled water. The sodium periodate solution was then added dropwise to the alginate–ethanol suspension under light exclusion with constant stirring (250 RPM). For the oxidation reaction, stirring was carried out for 6 h under the exclusion of light (i.e., the beaker was wrapped with aluminum foil). The reaction was stopped with 5 ml ethylene glycol, and then stirring was continued for 30 min. The ADA was dialyzed for 7 days against double distilled water to remove any remaining sodium periodate using the dialysis system Spectra/Por (Repligen, Boston, MA, USA) with standard RC dialysis membranes (6–8 kD MWCO). The water was changed twice a day. After dialysis, the ADA was dried in a lyophilization FreeZone Plus (Labconco, Kansas City, MO, USA) for another seven days. A 5% w/v ADA-solution was prepared by dissolving ADA in double-distilled water and stirred at 250 RPM overnight.

GTFs were extracted from liquid culture using dialysis in phenylmethylsulfonyl fluoride-containing PBS buffer, following previously published methods^{28 (link),29 (link)}. Supernatants of liquid culture of S. mutans were placed in 12–14,000 MW cut-off membranes (Spectrapor™, Repligen, Waltham, MA, USA) and proteins were extracted after overnight dialysis, then quantified using Bradford assay. Extracted and recombinant GTF-C and GTF-B (obtained with established methods^{30 (link)}) were subjected to electrophoresis under non-reducing conditions in MOPS buffer. After electrophoresis, gels were washed twice with 2.5% Triton-X 100 (30 min each) under agitation, and incubated in 0.2 M sodium phosphate buffer (pH 6.8) containing 0.2% dextran, 5% sucrose and 50 µM of the different inhibitors at 37 °C for 48 h. Gels were then washed twice with DI water, and stained with Coomassie Blue R-250 0.1%, washed with 50% methanol until enough contrast was observed against the background by visual inspection. The gels were then imaged in transmission (ChemiDoc Imaging System, Bio-Rad Laboratories, Hercules, CA), and kept in 1% acetic acid.