5 aminomethyl fluorescein hydrochloride

GluCDs were covalently conjugated to 5-(aminomethyl)-fluorescein hydrochloride (Thermo Fisher Scientific, Life Sciences; Carlsbad, CA) via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) coupling chemistry (Millipore-Sigma; St. Louis, MO). First, the –COOH groups on GluCDs were activated by EDC and NHS. 30 minutes later, 5-(aminomethyl)-fluorescein was added to the mixture. The mixture was stirred overnight protected from light. At the end, –COOH group on GluCDs' surface and –NH₂ of the 5-(aminomethyl)-fluorescein form a stable amide bond. GluCDs conjugated to 5-aminomethyl-fluorescein (GluCD-F) was purified by dialysis against DI-water, allowing unreacted small molecules to escape from the dialysis membrane with 1 kDa MWCO. Fluorescence emission of the dialysis water was measured periodically to monitor the purification and to ensure no free fluorescein is remained mixed with the GluCD-F conjugate. Finally, the purified conjugate was lyophilized to yield powdered product. Successful conjugation was confirmed by UV/Vis, fluorescence, and FTIR-ATR spectroscopies.

OA, domoic acid (DA), MC-LR, brevetoxin (BTX), soybean trypsin inhibitor (STI), bovine serum albumin (BSA), N,N′-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC), fluorescein 5(6)-isothiocyanate (FITC), methanol, chloroform, dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA), Triton X-100, and trimethylamine were acquired from Sigma-Aldrich (Saint Louis, MO, USA). Ethylenediamine dihydrochloride and 5-(aminomethyl) fluorescein hydrochloride (AMF) were acquired from ThermoFisher Scientific (Waltham, MA USA). A 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB) substrate solution was purchased from Immunotekh (Moscow, Russia). Goat anti-mouse immunoglobulins labeled with horseradish peroxidase (GAMI–HRP) were obtained from Jackson Immuno Research Labs (West Grove, PA, USA). Monoclonal antibodies (MAbs) against OA (clone 7E1) were acquired from Santa Cruz Biotechnology (Dallas, TX, USA). All other compounds were analytically pure. For the ELISA test, polystyrene 96-well transparent microplates Costar 9018 from Corning Costar (Tewksbury, MA, USA) were used.