Pyrenes

A controlled pore glass support (CPG) derivatized with 2′-O-methyl-A, 2′-O-methyl-G, deoxythymidine, 5′,N-protected 2′-O-methylribo- (A, C, G, or U), 2′-O-TBDMS-ribo (A, C, G, or U) and deoxyribo (dT) phosphoramidites, 2-[2-(4,4’-dimethoxytrityloxy)ethylsulfonyl]ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (CPR, Chemical Phosphorylation Reagent) were purchased from Glen Research Inc. (Sterling, VA, USA). Propargylamine, 1,6-diaminohexane, (pyrene-1-yl-methyl)amine hydrochloride, p-anisic (4-methoxybenzoic) acid, N,N′-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole hydrate (HOBt), N,N-diisopropylethylamine (DIPEA), α-GalNac-azide, and 1 M TBAF solution in THF were purchased from Sigma-Aldrich (St. Louis, MO, USA), N-Boc-1,6-diaminohexane hydrochloride and ethoxytrimethylsilane were obtained from Alfa Aesar (Heysham, UK); cholesterol chloroformate and oleylamine were obtained from Acros Organics (Geel, Belgium); 3-amino-1-propanol, triphenylphosphine (PPh₃), and 2,2′-dipyridyl disulfide ((PyS)₂) were obtained from Fluka (St. Louis, MO, USA). FAM-NHS, FAM-azide, 10 mM Cu(II)-TBTA Stock in 55% DMSO, and ascorbic acid were purchased from Lumiprobe (Moscow, Russia). All solvents (THF, DMSO, CH₃CN (various vendors)) were dried by 3 Å molecular sieves or by distillation and stored over CaH₂. Small molecule ligands were analyzed by thin-layer chromatography (TLC) using DC-Alufolien Kieselgel 60 F₂₅₄ plates (Merck, Darmstadt, Germany) at 254 nm ultraviolet light.

The membrane fluidity of SH-SY5Y cells was measured using the lipophilic pyrene probe pyrene decanoate (PDA) in the Membrane Fluidity Kit (ab189819, Marker Gene Technologies, Inc., Eugene, OR, USA). SH-SY5Y cells were seeded onto collagen-coated 96-well black plates at 1 × 10⁶ cells/mL; treated with Aβo, Aβo + curcumin, or Aβo + GT863 for 3 h; and then stained with a fluorescent lipid reagent containing PDA. When PDA forms excimers from monomers in spatial motion, the emission spectrum of PDA shifts to red. The ratio of fluorescence emission at 400 and 470 nm from 360 nm excitation (monomer-to-excimer ratio) was measured using a Spectra Max i3 (Molecular Devices) and was used as an estimate of membrane fluidity.

Absolute ethyl alcohol, benzoic acid, HNO₃, KCl, K₃Fe(CN)₆, K₄Fe(CN)₆·3H₂O, and K₂S₂O₈ were purchased from Aladdin Co. Ltd. (Shanghai, China). Lysozyme, Nickel-nitrilotriacetic acid (Ni-NTA), Sepharose, Phenylmethyl sulfonyl fluoride (PMSF), and color mixed protein marker PR1920 (11–245KD) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). N, N-Dimethylformamide (DMF), dichloromethane, 1,3,6,8-Tetra(4-carboxyphenyl)pyrene (H₄PTPA), and Zirconyl chloride octahydrate (ZrOCl₂·8H₂O) were procured from Macklin Biochemical Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA), N-Hydroxy succinimide (NHS), and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were ordered from Sigma Life Science Co. Ltd. (Shanghai, China). g-CN was received from Nanjing XFNANO Technology Co., Ltd. (Nanjing, China). OTA-Apt was synthesized from Sangon Biotech Co., Ltd. (Shanghai, China), the OTA aptamer with -NH₂ modification (5′-NH₂GATCGGGTG TGGGTGGCGTAAAGGGAGCATCGGACA-3′). Ochratoxin B (OTB), ochratoxin C (OTC), aflatoxin B1 (AFB1), fumonisins B1 (FB1), zearalenone (ZEN), and deoxynivalenol (DON) were obtained from Pribolab Co., Ltd. (Qingdao, China). The coffee sample was obtained from the supermarket in Haikou. All solutions were prepared by deionized water obtained from the Millipore water purification system (18.2 MΩ cm⁻¹, Milli-Q). Other reagents were analytical grade without further purification. The size and morphologies of the g-CN and NU-1000(Zr) nanocomposites were recorded by scanning electron microscope (SEM, FEI Helios G4 CX) and transmission electron microscopy (TEM, FEI Talos F200s). The cyclic voltammetry (CV) experiments were carried out on the CHI660e electrochemistry workstation (Shanghai CH Instruments, Shanghai, China) in 0.1 M KCl containing 5 mM [Fe(CN)₆]³⁻ and the ECL signals were taken by a model MPI-E electrochemiluminescence analyzer (Xi’An Remax Electronic Science & Technology Co. Ltd., Xi’an, China) in 0.1 M PBS containing 20 mM K₂S₂O₈ and 0.1 M KCl. The standard three-electrode system was used in the experiment, with the modified glass carbon electrode (GCE) as the working electrode, the platinum wire as the auxiliary electrode, and the saturated calomel electrode (SCE) as the reference electrode for CV, the Ag/AgCl (3.5 M KCl) as the reference electrode for ECL.