• Human CRP Duoset kit (R & D Systems, cat. no. DY1707E) !CAUTION Store reconstituted Ab and antigen at 2-8 °C, if they are to be used within a month. Otherwise, prepare aliquots and store at -20 °C to -70 °C for up to 6 months. The kit comprises of
• Mouse anti-human CRP capture Ab (360 μg mL-1)
• Recombinant human CRP (90 ng mL-1)
• Biotinylated mouse anti-human CRP detection Ab (22.5 µg mL-1)
• Streptavidin-conjugated horseradish peroxidase (SA-HRP) !CAUTION Do not freeze. Store in the dark as streptavidin is light-sensitive.
The Human CRP Duoset kit’s components can also be purchased separately, i.e. human CRP Ab (cat. no. MAB17071), human CRP biotinylated antibody (cat. no. BAM17072) and recombinant human CRP (cat. no. 1707-CR).
• Blocker BSA in PBS (10X), pH 7.4, 10% (w/v) (Thermo Scientific, cat. no. 37525) CRITICAL Filter with 0.2 µm pore size filter paper prior to use to avoid contamination.
• Sulfuric acid (Aldrich, cat. no. 339741) !CAUTION Use personal protective equipment (PPE), such as chemical safety glasses, chemical-resistant shoes and lab coats, for handling. Handle only in a fume cabinet. Avoid skin contact as it is a strong corrosive agent and an irritant. In case of skin contact, wash immediately with acid neutralizers and seek medical advice as soon as possible.
• 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 DIW (18Ω), 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 DIW, see REAGENT SETUP.
• TMB substrate kit (Thermo Scientific, cat. no. 34021)
• TMB solution (0.4 g L-1) !CAUTION Skin, eye and lung irritant. In case of skin contact, wash with plenty of water. CRITICAL Maintain the TMB to peroxide ratio as 1:1 as it is critical for color development.
• Hydrogen peroxide solution (containing 0.02 % v/v H2O2 in citric acid buffer) (Thermo Scientific). !CAUTION Use PPE and work in a safety cabinet or fume cupboard. It is a strong oxidizing agent, harmful if swallowed, and caused severe risk of damage to eyes. In case of contact, rinse immediately with plenty of water and seek medical attention.
• KOH pellets (99.99%), semiconductor grade (Sigma Aldrich, cat. no. 306568) !CAUTION Use PPE and handle in a safety cabinet. Avoid contact with skin and eyes as it can cause severe burns. CRITICAL The concentration of KOH must be 1% (w/v) in autoclaved DIW. Higher concentrations may affect the surface properties, thereby leading to decreased binding of capture antibodies.
• 3-aminopropyltriethoxysilane (3-APTES) (Sigma Aldrich, cat. no. A3684) !CAUTION Use PPE and handle in a safety cabinet. Avoid contact with skin and eyes as it is a skin and eye irritant, and highly toxic to kidney. CRITICAL Prepare in autoclaved DIW, see REAGENT SETUP.
• Human whole blood (HQ-Chex level 2) (Streck, cat. no. 232754) 180 day closed-vial stability and 30 day open-vial stability.
• Human serum (CRP free) (HyTest Ltd., cat. no. 8CFS)
• 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (Thermo Scientific, cat. no. 22981) !CAUTION Use PPE and handle inside a fume cupboard as it is an irritant. Being hygroscopic, it absorbs moisture that leads to loss of its activity. Equilibrate to room temperature (RT) before opening the container. CRITICAL Store at recommended temperature (-20 °C). Reconstitute in 0.1M MES, pH 4.7, see REAGENT SETUP.
• Graphene nanoplatelets (diameter 5 µm) (Cheap Tubes Inc., cat. no. Grade 2 Graphene Nanoplatelets)
• Recombinant human serum albumin (HSA), human fetuin A (HFA), human lipocalin 2 (LCN2), interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α (RnD Systems)
• Deionized water (18 Ω, DIW). (Millipore, Direct-Q®3 Water Purification System)
• 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 version 11.2 (Systat)
PBS. Add a BupH PBS pack to 100 mL of autoclaved DIW, dissolve well and adjust the volume up to 500 mL using autoclaved DIW. Each pack makes 500 mL of PBS at pH 7.2, which can be stored at RT for a week and up to four weeks at 4ºC
MES. Add a BupH MES pack to 100 mL of autoclaved DIW, dissolve well and adjust the volume up to 500 mL using autoclaved DIW. Each pack makes 500 mL of MES at pH 4.7, which can be stored at RT for up to two weeks.
APTES. Reconstitute the commercially supplied APTES solution (99% purity) in autoclaved DIW to make an effective 0.25% (v/v) solution. Prepare a fresh solution for each DIA run.
EDC. Each pack contains 25 g EDC. Reconstitute in 0.1M MES buffer, pH 4.7, at a concentration of 8 mg mL-1. Aliquots can be stored for six months at -20 °C.
Binding buffer. 0.1% BSA in PBS, pH 7.4.
Washing buffer. 0.05% Tween® 20 in PBS, pH 7.4 (PBST).
GNPs. GNPs (1 mg) was mixed with 1 mL of 0.25% APTES followed by sonication for 1 h.
EDC-activated anti-human CRP Ab. Dissolve 0.4 mg EDC in 100 µL of 0.1M MES, pH 4.7. Incubate 990 μL of the anti-human CRP Ab (4 μg mL-1) with 10 μL of EDC (4 mg mL-1) solution for 15 min at RT. CRITICAL STEP The concentration of EDC is important for optimal cross-linking. Use the recommended concentration of EDC. ?TROUBLESHOOTING
Anti-CRP capture Ab-bound GNPs. The EDC-activated Ab was mixed with GNPs (1 mg mL-1) in 0.25% APTES in the ratio of 1:1 (v/v). Thereafter, the anti-human CRP Ab solution (2 µg mL-1, 0.5 mg mL-1 GNPs and 0.125% APTES) was added to the MTP wells and incubated for 30 min at RT. The resulting wells were then washed five times with PBST.
Biotinylated anti-CRP detection Ab conjugated to SA-HRP. Biotinylated anti-CRP detection Ab conjugated to SA-HRP was prepared by adding 1 µL of biotinylated anti-CRP detection antibody (0.5 mg mL-1) to 1 µL of SA-HRP to 2998 µL of the binding buffer followed by 20 min of incubation at RT. As a result, the concentration of biotinylated anti-CRP detection Ab used was 0.17 µg mL-1, while SA-HRP dilution employed was 1:3000.
CRP spiked diluted human whole blood or serum. The CRP spiked samples (0.3-81 ng mL-1) were prepared by mixing the desired CRP concentrations in 1:100 diluted human whole blood/serum.
EDTA plasma samples from anonymized patients. The EDTA plasma samples from anonymized patients were diluted 1:1000 and 1:4000 in the binding buffer. The resulting CRP concentration in these samples falls within the linear range of the DIA, thereby enabling the detection of an entire pathophysiological concentration range of human CRP.