Ddh2o

SupT1 and SupT1-R5 cells (1 × 10⁴ cells per sample) were pelleted and resuspended in 20 μL cold RPMI-1640 with 0.4% FBS and 6 μg/mL OKT4-Alexa Fluor 647, with or without 1 μg/mL HIV_BaL gp120 (NIH AIDS Reagents Program). Cells were incubated on ice for 60 min before being diluted in 10 mL cold serum-free RPMI-1640 and centrifuging at 300 RCF for 6 min at 4 °C. The supernatants were carefully aspirated, and the cells resuspended and washed twice in 10 mL cold serum-free RPMI-1640. The cells were then resuspended in 60 μL cold serum-free RPMI-1640 and allowed to settle on PLL-coated coverslips (100 μg/mL in ddH₂O, Sigma-Aldrich, Dorset, UK, P8920), on ice, for 40 min. Control samples were transferred directly to cold 4% PFA. Treated samples were transferred to 37 °C serum-free RPMI-1640 for 1 min, before returning to 1 mL cold serum-free RPMI-1640 for 5 min to rapidly cease cells’ reaction to treatment. The cells were then fixed with cold 4% PFA for 10 min, before warming to 37 °C over 20 min. Samples were washed five times in PBS and stored in PBS until mounting for imaging.

Bacteria were standardized (1 × 10⁷ cfu/mL) in artificial saliva (AS), which contained the following constituents, as described previously
[11 (link)]. This included porcine stomach mucins (0.25% w/v), sodium chloride (0.35 w/v), potassium chloride (0.02 w/v), calcium chloride dihydrate (0.02 w/v), yeast extract (0.2 w/v), lab lemco powder (0.1 w/v), proteose peptone (0.5 w/v) in ddH₂O (Sigma, Poole, UK). Urea was then added to independently to a final concentration of 0.05% (v/v). To initiate multispecies biofilm development the pioneer species S. mitis biofilm were first formed for 24 h in 5% CO₂ on 13 mm diameter Thermanox™ coverslips within 24 well plates (Corning, NY, USA). The supernatant was then removed and F. nucleatum added, which was incubated anaerobically at 37°C for a further 24 h. The supernatant was removed and P. gingivalis and A. actinomycetemcomitans added to the dual species biofilm, which was incubated anaerobically at 37°C for a further 4 days, replacing the AS daily to produce a mixed four species biofilm (Figure 
1A).

A chromatographic separation assay was performed using the Thermo Ultimate 3000 platform coupled with an ACQUITY UPLC^® HSS T3 column (2.1 × 150 mm × 1.8 μm; Waters Co. Ltd., Milford, MA, USA). The temperature of the automatic sampler was set to 8°C. Gradient elution of analytes was carried out with 0.1% formic acid (TCI Co. Ltd., Tokyo, Japan), inddH2O (Millipore), and 0.1% formic acid in acetonitrile (ThermoFisher Scientific, USA) or 5.0 mM ammonium formate (Sigma) in ddH2O and acetonitrile at a flow rate of 0.25 mL/min. Injection of 2.0 μL of each sample was performed after equilibration.

After the Neuro-2a cells were cultured in the C, N and B conditions for 24 h, the cells were stained with PI (1 μg/mL, dissolved with sterile ddH2O, Sigma-Aldrich, St. Louis, MO, USA) solution for 1 h. Fluorescence was monitored at 200× magnification using microscopy (Olympus, Tokyo, Japan). To compare the fluorescence intensity in Neuro-2a cells among the groups, Image J software (Version 1.52t, NIH, Bethesda, MD, USA) was used to quantify the fluorescence intensity in a single cell. In brief, after 4–6 images (each image representing one biological replicate) of each group were taken at random locations, three different cells representing technical replicates in each image were randomly selected to determine the level of fluorescence with the ImageJ software.

Graphene oxide was prepared from a natural graphite powder (Bay carbon Inc., Bay City, MI, USA) using a modified Hummers’ method [10 (link),37 (link),38 (link),39 (link),40 (link)]. Graphite (8.5 M) and NaNO₃ (0.6 M) (Merck & Co., Kenilworth, NJ, USA) were mixed with H₂SO₄ (FUJIFILM Wako Chemicals USA Inc., Richmond, VA, USA). KMnO₄ (2.0 M) (Fisher Scientific, Hampton, NH, USA) was slowly added with continual stirring at 35 °C overnight. Subsequently, deionized water (ddH₂O) was gradually added and continually stirred. Adding H₂O₂ (Sigma Aldrich Co., St. Louis, MO, USA) was the method used to terminate the reaction. Washing and centrifugation with ddH₂O several times were carried out, and the graphene oxide was collected. The as-prepared graphene oxide was placed in a tube furnace and heated to 400–600 °C [400 °C and 600 °C for amino-N-GQD (4.9%) and amino-N-GQD (6.2%) of the N(1s)/C(1s) ratio determined by XPS, respectively] in the presence of ammonia for 4–6 h. N-GQDs were subsequently obtained following the same procedure. The as-prepared N-GQDs were mixed with ammonia (Sigma Aldrich Co., St. Louis, MO, USA), stored in a Teflon-lined stainless-steel autoclave, and reacted at 180 °C for 5 h. The resulting mixture was washed with ddH₂O, centrifuged several times, and subsequently dried in an oven at 50 °C overnight. Eventually, amino-N-GQDs were obtained.