Gold nanoparticles

SCC-Ag was obtained from RANDAX Life Sciences (Malaysia), anti-SCC-Ag antibody was purchased from Next Gene (Malaysia), ethanolamine, (3-aminopropyl)triethoxysilane (APTES), ethanol, PBS (phosphate buffered saline), 16-mercaptoundecanoic acid, and human serum were obtained from Sigma-Aldrich (USA), and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were procured from GE Healthcare (USA). Gold nanoparticles with a size of 30 nm were purchased from Sigma-Aldrich (USA). α-Fetoprotein and CYFRA 21-1 were from MyBioSource (USA). All other reagents used were of analytical grade.

All reagents were of analytical grade and were used without further purification. Distilled water was used throughout the experiments. Pure grade of naloxone hydrochloride 99.8% and Narcan® ampoules, each ampoule (1 mL) claimed to contain 400 μg mL⁻¹ naloxone hydrochloride were kindly supplied by Bristol Myer Squibb Co., Egypt. 5.0×10⁻⁴ mol L⁻¹ luminol (Sigma Chemical Co.) stock solution was prepared in 100 mL of 1.0×10⁻² mol L⁻¹ sodium hydroxide (WinLab). Potassium ferricyanide (WinLab) was used to prepare 1.0×10⁻² mol L⁻¹ solution by dissolving 0.33 g in 100 mL distilled water. Gold nanoparticles 1.0×10⁻¹ mol L⁻¹ were purchased from (Sigma-Aldrich -Germany). Urine samples were obtained from healthy volunteers and serum samples (Multi-Serum Normal, Randox Laboratories, UK) were obtained from commercial sources.

Titanium foil with a chemical purity of 99.7%, ammonium fluoride with chemical purity ≥98%, phosphate buffered saline (0.01 M PBS, pH 7.4), ethylene glycol (assay 99.8%), gold (III) chloride hydrate HAuCl₄∙3H₂O (assay 99.995%), gold nanoparticles (10 nm stabilized suspension in 0.1 mM PBS), bovine serum albumin (BSA, purity ≥ 98%), were purchased from Sigma-Aldrich (St. Louis, MO, USA). All of the solutions were prepared from Milli-Q water.

Before image capture, samples were incubated with 0.01% poly-L-lysine (Sigma) for 10 min and then incubated with 150 nm gold nanoparticles (Sigma) diluted 1:25 in PBS for use as fiducial markers as described previously⁶⁴ . Calibration images of the point spread function (PSF) over a 4 µm range in 50 nm steps were taken from selected fiducial markers. Data was collected in the presence of oxygen scavenging buffer consisting of glucose oxidase and catalase (10 and 50 U, respectively; Sigma), 12.5 mg/ml D-glucose, and 1 mM 2-mercaptoethylamine (Sigma) in PBS (pH 8.0). Oxygen scavenging buffer was replenished every hour with a freshly prepared solution. Fluorophores were stochastically activated under wide-field illumination with 642 nm and 405 nm lasers at 50 mW and up to 1.6 µW, respectively. Multiple data sets of 11,000 raw images were acquired at a frame rate of 20 Hz and camera gain of 100. Fluorescent events were localised in x-y and z by cross-correlation with the PSFs captured in the calibration file, and corrected for drift by tracking the gold nanoparticles using palm3d software^{34 (link)} (see https://github.com/AndrewGYork/palm3d). Localisations with a cross-correlation with the calibration PSF below 0.4 were rejected^{34 (link)}.