Micrococcus lysodeikticus cells

Assays were carried out in triplicate, using flat-bottomed 96-well untreated ELISA plates (Greiner). OBodies suspended in PBS pH 7.4 were first diluted 1∶2 serially over 11 steps, starting with a final assay concentration of 50 µM. A 25 µL aliquot of each OBody dilution was either added to 25 µL hen egg-white lysozyme (Sigma) at 648 nM (162 nM final concentration in assay) or 25 µL PBS pH 7.4 and allowed to equilibrate at room temperature for 15 min. To each OBody dilution, a 50 µL aliquot of substrate solution containing inactivated Micrococcus lysodeikticus cells (Sigma, M3770) suspended in PBS pH 7.4 was added to a final concentration of 0.4 mg/mL and plates were incubated at room temperature for 45 min before reading the absorbance at 450 nm in a BMG Fluostar Optima plate reader. The data were normalised and processed using Prism v5.04 (Graphpad) software and non-linear regression performed using the following model [Y = Bottom+(Top-Bottom)/(1+10<$>\vskip -3 \raster(60%)="rg1"<$>((LogIC₅₀−X)*HillSlope))] to obtain IC₅₀ values.

Plasma lysozyme activity was determined by a turbidimetric assay utilizing lyophilized Micrococcus lysodeikticus cells (Sigma, Rowville, VIC, Australia) using a method modified from Cha et al. (2008) (link). Lysozyme from chicken egg white (Sigma, Rowville, VIC, Australia) was used as a standard. In a 96 well plate, 15 µL of plasma samples diluted 1:40 in 0.02 M sodium citrate buffer were added to 150 µL of M. lysodeikticus suspended in 0.02 M sodium citrate buffer at a concentration of 0.2 mg/mL. The absorbance was immediately measured at 450 nm, and subsequent measurements were taken every 5 min for 60 min. A unit of lysozyme activity was defined as the quantity of plasma required to reduce absorbance by 0.001/min (111).

Bovine heart cytochrome c, bovine carbonic anhydrase b and dried Micrococcus lysodeikticus cells were purchased from Sigma. Hen egg white lysozyme (20000 units/mg), yeast cytochrome c, reduced and oxidized glutathione (highly pure), urea (highly pure) and guanidine hydrochloride (highly pure) were purchased from Shanghai Sangon Biological and Technological Service Co., Ltd. All other reagents were of analytical grade without further purification, and all solutions were prepared in bi-distilled water and passed through a 0.22 µm filter.

The lysozyme activity was assessed using the previously described method [52 ,53 ]. Briefly, a turbidimetric assessment utilizing lyophilized Micrococcus lysodeikticus cells (Sigma) was performed. M. lysodeikticus (150 µL) with a concentration of 0.2 mg/mL (in 0.02 M sodium citrate buffer, pH = 5.5) was added to 15 µL sera in a 96-well U-bottom microtiter plate. The initial optical density (OD) was detected at 450 nm instantly after adding M. lysodeikticus, and then, the final OD was measured after incubation for 1 h at 37 °C. Lyophilized hen egg-white lysozyme (Sigma) was used to develop a standard curve. The lysozyme levels were expressed as units·mL⁻¹, with a decrease in absorbance of 0.001 min⁻¹ representing one unit.

Lyophilized chicken egg white lysozyme (EC 3.2.1.17) was purchased from Inovatech, Inc. (Abbotsford, BC, Canada), and Micrococcus lysodeikticus cells, obtained from Sigma-Aldrich Corporation (St. Louis, MO) as salt-free and dry powder, were used without further purification. Carboxyl single-walled carbon nanotubes (SWCNT-COOH) with outer diameter of 1–2 nm were purchased from MKnano, Canada. MES [2-(N-morpholino) ethane sulfonic acid] buffer, N-ethyl-N’-(3-(dimethyl amino) propyl) carbodiimide hydrochloride (EDC), Tris-hydroxymethyl aminomethane (Tris), N,N methylenebisacrylamide (Bis), acrylamide, sodium dodecyl sulfate, ammonium persulfate, tetramethylethylenediamine (TEMED), 2-mercaptoethanol(2ME), 3,3–5,5 tetrabromophenolsulfonphthalein (Bromophenol Blue) and all other chemicals were purchased from Sigma-Aldrich Corporation and used as received.