Sodium borate buffer

4-Dimethylamino-cinnamaldehyde (DMACA), dimethyl sulfoxide (DMSO), 2,2-diphenyl-1-picrylhydrazyl (DPPH), formic acid, melanin from Sepia officinalis, n-butanol, phosphate buffer, potassium ferricyanide, potassium permanganate, potassium persulfate, potassium phosphate buffer, sodium borate buffer, sodium carbonate, sodium hydroxide, sodium potassium tartrate, and sulfuric acid was purchased from Sigma Chemical Company (St. Louis, Missouri, USA). Analytical-grade acetic acid, acetone, acetonitrile, chloroform, ethanol, ethyl acetate, hydrochloric acid, and methanol were purchased from Honeywell (Seelze, Hanover, Germany). Deuterated dimethyl sulfoxide (DMSO), used in NMR studies, was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). All other chemicals and reagents were of analytical grade and were used without purification.

Conjugation was performed as previously described [17 ]. Shortly, m11B6 (provided by the University of Turku, Finland) in 0.07 M sodium borate buffer (Sigma Aldrich, St Louis, MO, USA), pH 9.2, was concentrated on an Amicon Ultra-2 centrifugal filter, 2 mL, 100 K, (Millipore, Billerica, MA, USA) and conjugated at 40 °C with the chelator CHX-A''-DTPA (Macrocyclics, Dallas, TX, USA) in a chelator to antibody molar ratio of 3:1 for 4 h. The reaction was terminated and CHX-A''-DTPA-m11B6 was separated from free chelate by size-exclusion chromatography using a NAP-5 column (GE Healthcare, Uppsala, Sweden), equilibrated with 20 mL of 0.2 M ammonium acetate buffer pH 5.5. The conjugated antibody was kept at −20 °C for labeling.

The hybridization chain reactions were validated using gel electrophoresis herein. The HCR firstly took place in reaction tubes under 1× PBS with 0.5 M NaCl (Sigma-Aldrich, USA) under room temperature. Such salt concentration is found to optimize the rate of reaction. Further increase of salt concentration up to 1× PBS with 1 M NaCl is found to significantly reduce the reaction rate despite the seemingly beneficial charge screening effect of higher ionic strength. This is likely due to the presence of a self-stable target sequence under higher salt concentrations. The H1 and H2 were firstly mixed in equal concentration, then the target strand was added into the solution for 30 min reaction time. The final concentration of H1 and H2 was 5 μM, and the concentration of the target was varied to demonstrate the HCR, as suggested by the first work reported by Dirk et al. [1 (link)]. The running gel for electrophoresis was made of 2% agarose in 1× sodium borate buffer (1× SB buffer) (Sigma-Aldrich, USA). The electrophoresis was done with a 100-V driving voltage (with MAJOR SCIENCE MP-100) at room temperature, using a 1× SB running buffer. The fluorescent dye used for imaging was Safe Green (Hycell, Taiwan). The concentrations of oligonucleotides used in gel electrophoresis were set to provide better fluorescence imaging results.

Calcium accumulation on materials has been identified as a potential factor to act as nucleation for osteogenic mineralisation [28] (link). Therefore, the potential of calcium accumulation of PEDOT:PSS scaffolds was assessed by incubating cell-free scaffolds in culture media. Specifically, four different conditions were chosen: dry, as produced scaffolds (ctrl), serum incubated scaffolds (d4 FBS), and scaffolds incubated in proliferation media for 1 or 7 days (d1, d7) followed by differentiation media for 21 days, to mirror cell experiments (d28).
PEDOT:PSS scaffolds were assessed for mineral deposition based on Ca²⁺ complexation with o-CPC. Scaffolds were harvested, rinsed in PBS and frozen in 0.1 v/v% triton-X at −20 °C.
After three freezing / thawing cycles, scaffolds were incubated in 0.75 M acetic acid and 0.1 v/v% triton-X supernatant at a 1:1 ratio for 6 h. The acidic solution with dissolved calcium was then incubated with a 0.01 w/v% o-cresolphtalein complexone solution (o-CPC, Sigma, St. Louis, MA, USA) in 0.25 M sodium borate buffer (pH 10, Sigma, St. Louis, MA, USA). Absorbance was measured at 570 nm (Perkin Elmer EnSpire Plate Reader, Waltham, MA, USA) and quantified with a Ca²⁺ standard curve from 1 M CaCl₂ solution (Sigma, St. Louis, MA, USA). Evaluation was accomplished in N = 3 individual experiments with three replicates each, resulting in n = 9 scaffolds.