4.1. Sample Preparation

Aβ_1–40 (purity > 95%) was synthesized in the Peptide Technology Facility of the Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne. The lyophilized peptide was dissolved at a nominal concentration of 1 mg/mL in 1,1,1,3,3,3-hexafluoroisopropanol and portioned, then the solvent was allowed to evaporate. The resulting peptide film was resuspended at 4 °C in 20% v/v 20 mM NaOH, 70% v/v ultrapure water (MilliQ; MERCK KGAA, Darmstadt, Germany), and 10% v/v 10 × phosphate-buffered saline (PBS; 10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl; Sigma). After centrifugation at 14,000× g for 15 min at 4 °C, the supernatant was retained and the peptide concentration was immediately determined using ε₂₁₄ = 74,925 M⁻¹cm⁻¹ [23]. A concentrated stock of ⁶⁵CuCl₂ was prepared by stirring ⁶⁵CuO (⁶⁵Cu, >99%; Cambridge Isotope Laboratories, Tewksbury, MA, USA) in concentrated HCl, evaporating excess HCl under heat, then adding ultrapure water. PBT2 was synthesized as previously described [24], and a 1 mM stock solution was prepared by solubilizing the hydrochloride salt directly in PBS.

From the above stock solutions, PBT2 and then ⁶⁵CuCl₂ were added to portions of Aβ_1–40 to achieve final molar Cu/PBT2/ratios of n:1:1 (n = 0.33–2.67) with [Aβ_1–40] = 200 μM. Control samples containing Cu/PBT2 0.5:1, Cu/PBT2 1:1, Cu/Aβ_1–40 1:1, and Cu/Aβ_1–40 2.5:1 were also prepared. Glycerol (10% v/v) was added to the Cu/PBT2 control samples to prevent formation of a concentrated solute phase upon freezing. Immediately after Cu addition, the final solution pH was measured using a micro-probe (Hanna Instruments, Villafranca Padovana, Italy) and adjusted using concentrated NaOH or HCl as required. Samples were transferred to quartz EPR tubes (SQ-707; ATS Life Sciences Wilmad, Vineland, NJ, USA) and snap-frozen in liquid nitrogen within two minutes of the Cu addition.