• Human Lipocalin-2/NGAL DuoSet ELISA kit (R & D Systems, cat. no. DY1184e). All the assay components in the kit were reconstituted in phosphate buffered saline (PBS, 0.1 M, pH 7.2) with 1% (v/v) bovine serum albumin (BSA), while PBST (PBS with 0.05% Tween 20) was used for washing steps. The buffers were made with ultrapure water (UPW). The kit comprises of
• Rat anti-human LCN2 capture antibody (360 μg mL-1) !CAUTION Store reconstituted antibody at 2-8°C for up to 60 days or at -20°C to -70°C for up to 6 months. Dilute to a working concentration of 2 μg mL-1 in 0.1 M PBS, pH 7.2.
• Recombinant human LCN2 (90 ng mL-1). !CAUTION Store reconstituted LCN2 at 2-8°C for up to 60 days or at -70°C for up to 6 months. Dilute to the dynamic concentration range of the assay in 0.1 M PBS, pH 7.2) with 1% BSA. Make the dilutions of human LCN2 in BSA-preblocked sample vials to minimize the analyte loss due to non-specific binding on sample tube surfaces25.
• Biotinylated goat anti-human LCN2 detection antibody (18 μg mL-1). !CAUTION Store reconstituted antibody at 2-8°C for up to 60 days or at -20°C to -70°C for up to 6 months. Dilute to a working concentration of 100 ng mL-1 in 0.1 M PBS, pH 7.2) with 1% BSA.
• Streptavidin conjugated to HRP. !CAUTION Store at 2-8°C in dark for up to 6 months after initial use as streptavidin is light-sensitive.
• Blocker BSA in PBS (10X), pH 7.4, 10% (w/v) (Thermo Scientific, cat. no. 37525) !CRITICAL To avoid microbial contamination in BSA, filter prior to use.
• Sulphuric acid (Aldrich, cat. no. 339741) !CAUTION Avoid skin contact as it is a strong corrosive agent and an irritant. Use personal protective equipment (PPE), i.e. chemical-resistant shoes, lab coats and chemical safety glasses, for handling. Handle only in a fume cabinet. In case of skin contact, wash immediately with acid neutralizers and seek medical advice as soon as possible.
• KOH pellets (99.99%), semiconductor grade (Sigma Aldrich, cat. no. 306568) !CAUTION Use PPE for handling and handle only in a fume cabinet as it can cause severe burns. Avoid contact with skin and eyes. CRITICAL The KOH concentration must be 1% (w/v) in autoclaved UPW as higher concentrations lead to surface defects and strongly impacts the antibody immobilization.
• APTES (Sigma Aldrich, Ireland, cat. no. A3684) !CAUTION Higher concentrations are highly toxic. Avoid contact with skin and eyes as it is a skin and eye irritant. Handle inside a fume cabinet. CRITICAL Prepare in autoclaved UPW, see REAGENT SETUP.
• GNPs (diameter 5 µm) (Cheap Tubes, Grade 2) !CAUTION Avoid inhalation. CRITICAL Mix 1 mg of GNPs with 1 mL of 0.25% APTES and disperse by keeping in an ultrasonic bath for 1 h before use.
• 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (Thermo Scientific, cat. no. 22981) !CAUTION Equilibrate to room temperature (RT) as it is hygroscopic and may lose activity upon water absorption. Handle inside a fume cabinet as it is an irritant. CRITICAL Store at -20°C. Reconstitute in 0.1 M MES, pH 4.7, see REAGENT SETUP.
• BupH Phosphate Buffered Saline Packs (0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2) (Thermo Scientific, cat. no. 18372) !CAUTION Avoid inhalation. CRITICAL Prepare in autoclaved UPW, see REAGENT SETUP.
• BupH MES Buffered Saline Packs (0.1 M MES [2-(N- morpholino)ethane sulfonic acid], 0.9 % (w/v) sodium chloride, pH 4.7) (Thermo Scientific, cat. no. 28390) !CAUTION Avoid inhalation. CRITICAL Prepare in autoclaved UPW, see REAGENT SETUP.
• TMB substrate kit (Thermo Scientific, cat. no. 34021). It comprises of
• TMB solution (0.4 g L-1) !CAUTION Skin, eye and lung irritant. In case of skin contact, wash with plenty of water. CRITICAL STEP Maintain the TMB:peroxide ratio as 1:1 as it is critical for the development of color.
• Peroxide solution (containing 0.02 % (v/v) H2O2 in citric acid buffer) (Thermo Scientific). !CAUTION It is a strong oxidizing agent, which is harmful if swallowed and carries a severe risk of damage to eyes. In case of contact, rinse immediately with plenty of water and seek medical attention as soon as possible. Wear PPE and work inside the safety cabinet or fume cabinet.
• Nunc microwell 96-well polystyrene plates, flat bottom (non-treated), sterile (Sigma Aldrich, cat. no. P7491)
• Eppendorf microtubes (1.5 mL; Sigma Aldrich, cat. no. Z 606340)
• SigmaPlot software bundle version 11.2 from Systat for curve plotting and assay analysis