2 2 azino bis 3 ethylbenzothiazoline 6 sulfonic acid diammonium salt

Manufactured by Merck Group

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2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt is a water-soluble chromogenic substrate commonly used in various enzymatic assays and immunoassays. It functions as an electron acceptor, undergoing oxidation to produce a colored product that can be measured spectrophotometrically.

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Lab products found in correlation

Gallic acid, by Merck Group (69 mentions) 2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (57 mentions) Potassium persulfate, by Merck Group (44 mentions) Quercetin, by Merck Group (37 mentions) Folin ciocalteu reagent, by Merck Group (27 mentions) Sodium carbonate, by Merck Group (27 mentions) Formic acid, by Merck Group (27 mentions) 6 hydroxy 2 5 7 8 tetramethylchroman 2 carboxylic acid trolox, by Merck Group (23 mentions) 6 hydroxy 2 5 7 8 tetramethylchroman 2 carboxylic acid, by Merck Group (23 mentions) Sodium acetate, by Merck Group (21 mentions) Trolox, by Merck Group (21 mentions) Sodium hydroxide (naoh), by Merck Group (21 mentions) Ascorbic acid, by Merck Group (20 mentions) 6 hydroxy 2 5 7 8 tetramethylchromane 2 carboxylic acid trolox, by Merck Group (20 mentions) Catechin, by Merck Group (19 mentions) Acetic acid, by Merck Group (19 mentions) 2 4 6 tris 2 pyridyl s triazine, by Merck Group (18 mentions) Methanol, by Merck Group (17 mentions) Folin ciocalteu s phenol reagent, by Merck Group (16 mentions) Chlorogenic acid, by Merck Group (15 mentions)

214 protocols using 2 2 azino bis 3 ethylbenzothiazoline 6 sulfonic acid diammonium salt

Synthesis of Stimuli-Responsive Hydrogels

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N-(3-Aminopropyl)methacrylamide
hydrochloride (APMA), N-isopropylacrylamide (NIPA), N,N′-methylenebis(acrylamide) (BIS), N,N,N',N'-tetramethylethylenediamine (TEMED),
ammonium persulfate
(APS), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt (ABTS), and ascorbic acid were purchased from Aldrich.
Sodium nitrate (NaNO₃) and sodium hydroxide were purchased
from POCh. NIPA was purified using recrystallization from a mixture
of toluene/hexane (30:70, v/v). All other chemicals were used as received.
The solutions were prepared using high-purity water obtained from
a Milli-Q Plus/Millipore purification system (water conductivity of
0.056 μS cm^–1).

Kaniewska K., Marcisz K, & Karbarz M. (2023). Temperature-Modulated Changes in Thin Gel Layer Thickness Triggered by Electrochemical Stimuli. Langmuir, 39(6), 2398-2407.

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Electrochemical Characterization of Natural Organic Matter

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Aldrich (Milwaukee, MI): 2,2 ′ -azino-bis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, 98%), diquat dibromide monohydrate (DQ, analytical standard), dimethyl sulfoxide (DMSO, 99.9%), tetrabutylammonium hexauorophosphate (TBAFP, ACS reagent grade), potassium chloride (KCl, 99%), potassium phosphate monobasic (KH 2 PO 4 , 99%), potassium phosphate dibasic (K 2 HPO 4 , 99%), 5-hydroxy-1,4-naphthoquinone (juglone, 97%), 1,2naphthoquinone-4-sulfonic acid (o-NQS, 97%). VWR chemicals (Radnor, PA): L-ascorbic acid (ACS grade). Tokyo Chemical Industry (TCI, Tokyo, Japan): disodium anthraquinone-2,6disulfonate (AQDS, 98%). All NOM samples used in this study, including each sample's source, elemental composition, and class are provided in Table S1. † Deionized (DI) water was further puried using a Milli-Q Biocel pure water system ($18.2 MU cm at 25 °C; Millipore Sigma, St. Louis, MO, USA). All reagents were used without additional purication.

Cao H., Pavitt A.S., Hudson J.M., Tratnyek P.G, & Xu W. (2023). Electron exchange capacity of pyrogenic dissolved organic matter (pyDOM): complementarity of square-wave voltammetry in DMSO and mediated chronoamperometry in water. Environmental science. Processes & impacts, 25(4).

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Fluorescent Enzyme Characterization and ATP-PDDA Coacervates

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All reagents were purchased through commercially available sources: dextran (DEX: Mw 500,000), polyethylene glycol (PEG: Mw 35,000), horseradish peroxidase (HRP: 150 units mg⁻¹), glucose oxidase (GOx: 15 units mg⁻¹), dextranase (17.5 and 175 units mg⁻¹), fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC), ampliflu™ red, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 3,3’-diaminobenzidine (DAB), o-phenylenediamine (oPD), 2-(N-morpholino)ethanesulfonic acid (MES) monohydrate, 3-morpholinopropane-1-sulfonic acid (MOPS), sodium hydroxide (6 M NaOH), hydrochloric acid (1M HCl), and Pur-A-Lyzer Maxi dialysis kit (Mw cutoff 12,000) were from Millipore-Sigma. FITC-DEX (Mw 500,000) was from TdB Consultancy AB. Food colorings (blue, green, red, and yellow) were from ESCO Foods. 35 mm glass-bottom dishes (No.0) were from MatTek Corp. Flat-bottom 24-well plates were from Corning. Fluorescent labelling of enzymes and characterization of ATP-PDDA coacervates were conducted using a previously reported protocol^{26 (link)} and described in the supporting information.

Kojima T, & Takayama S. (2018). Membrane-less compartmentalization facilitates enzymatic cascade reactions and reduces substrate inhibition.. ACS applied materials & interfaces, 10(38), 32782-32791.

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Antioxidant Activity Evaluation Methods

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The following chemicals and enzymes (catalogue numbers in parentheses) were purchased from Millipore-Sigma (St. Louis, MO, USA): 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, #648471), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, #A1888), 2,2-diphenyl-1-picrylhydrazyl (DPPH, #D9132), potassium persulfate (#216224), xanthine (#X0626), L-glutathione reduced (#G4251), xanthine oxidase from bovine milk (#X1875), Cu/Zn superoxide dismutase from bovine erythrocytes (#S5395), and horseradish peroxidase (#P8375). Amplex^TM UltraRed Reagent (#A36006) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Other chemicals (e.g., KCl, K₂HPO₄, MgCl₂, etc.) were obtained at high purity (≥99%) from Millipore-Sigma.
Mdivi-1 was purchased from Millipore-Sigma (#M0199) and prepared as a 50 mM stock in dimethyl sulfoxide (DMSO). A 1:500 dilution of DMSO (0.2%), equivalent to the highest tested Mdivi-1 concentration (100 μM), was used as the vehicle control for all experiments.

Bordt E.A., Zhang N., Waddell J, & Polster B.M. (2022). The Non-Specific Drp1 Inhibitor Mdivi-1 Has Modest Biochemical Antioxidant Activity. Antioxidants, 11(3), 450.

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Antioxidant Compounds Analysis Protocol

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Folin–Ciocalteau reagent and Iron (III) chloride hexahydrate (FeCl₃·6H₂O) were acquired from Dinâmica (São Paulo, Brazil). Methanol (Merck, Darmstadt, Germany) and formic acid (Panreac, Barcelona, Spain) were HPLC grade. 2,4,6-Tris(2-piridil)-s-triazina (TPTZ), 2,2-difenil-1-picril-hidrazil (DPPH), 6-Hidroxi-2,5,7, 8-tetrametilcromano-2-carboxylic acid (Trolox), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium persulfate, gallic acid, catechin hydrate, chlorogenic acid, caffeic acid, ellagic acid hydrate, and e quercetin were supplied by Sigma-Aldrich (St Louis, MO, USA). Potassium carbonate and Deionized water were purchased from Synth (Diadema, Brazil) and obtained from a Milli-Q (Millipore, Billerica, MA, USA) purification unit, respectively.

Macedo M.C., Correia V.T., Silva V.D., Pereira D.T., Augusti R., Melo J.O., Pires C.V., de Paula A.C, & Fante C.A. (2023). Development and Characterization of Yellow Passion Fruit Peel Flour (Passiflora edulis f. flavicarpa). Metabolites, 13(6), 684.

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Standardized Compounds for Antioxidant Assays

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The standard compounds, including cyanidin-3-O-galactoside (90% purity), cyanidin-3-O-glucoside (95% purity), cyanidin (95% purity), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (98% purity), 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (98% purity), 2,9-dimethyl-1,10-phenanthroline (Neocuproine) (99% purity), potassium persulfate (99% purity), acetonitrile, formic acid, ethanol, methanol, and dimethyl sulfoxide (DMSO), were obtained from Sigma-Aldrich (Darmstadt, Germany). cyanidin-3-O-galactoside and cyanidin-3-O-arabinoside standards were bought from Polyphenols (Sandnes, Norway). HCl, NaNO₂, H₂O₂, and CuCl₂ were purchased from Merck (Darmstadt, Germany). Fetal bovine serum (FBS), Lonza, Dulbecco's Modified Eagle Medium (DMEM), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Lonza Group Ltd. (Basel, Switzerland). Glutamine, penicillin and streptomycin, and amphotericin were purchased from Sigma Chemical Co. (St. Louis, MO). The water used for experiments was treated in a Milli-Q water purification system.

Rugină D., Diaconeasa Z., Coman C., Bunea A., Socaciu C, & Pintea A. (2015). Chokeberry Anthocyanin Extract as Pancreatic β-Cell Protectors in Two Models of Induced Oxidative Stress. Oxidative Medicine and Cellular Longevity, 2015, 429075.

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Antioxidant Capacity Assay Reagents

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1,1-diphenyl-2-picrylhydrazyl radical (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), Folin & Ciocalteu’s phenol reagent, 2,4,6-tris-(2-pyridyl)-s-triazine (TPTZ), (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), potassium persulfate (K₂S₂O₈), and gallic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium carbonate (Na₂CO₃), iron (III) chloride hexahydrate (FeCl₃ 6H₂O), hydrochloric acid (HCl), methanol (MeOH), sodium phosphate monobasic (NaH₂PO₄) and sodium phosphate dibasic (Na₂HPO₄) were purchased from Merck (Darmstadt, Germany).

Touma J., Navarro M., Sepúlveda B., Pavon A., Corsini G., Fernández K., Quezada C., Torres A., Larrazabal-Fuentes M.J., Paredes A., Neira I., Ferrando M., Bruna F., Venegas A, & Bravo J. (2020). The Chemical Compositions of Essential Oils Derived from Cryptocarya alba and Laurelia sempervirens Possess Antioxidant, Antibacterial and Antitumoral Activity Potential. Molecules, 25(23), 5600.

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ABTS Antioxidant Assay Protocol

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The ABTS assay was performed following the method described by Contreras et al. 2011 [15 (link)]. One hundred microliter of sample (diluted appropriately with water) was mixed with 1 mL of ABTS⁺ solution, which was prepared with ABTS standard (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, lot slbp9592v, sigma-Aldrich, St. Lois, USA) diluted in ethanol. The change in coloration was read after 30 min at 730 nm in a spectrophotometer (UV-1700, Shimadzu). The results were expressed as trolox equivalents (TE) (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid to 97%, lot stbb6668, sigma-Aldrich, St. Lois, USA) or TE μmol /L.

Quintero Quiroz J., Naranjo Duran A.M., Silva Garcia M., Ciro Gomez G.L, & Rojas Camargo J.J. (2019). Ultrasound-Assisted Extraction of Bioactive Compounds from Annatto Seeds, Evaluation of Their Antimicrobial and Antioxidant Activity, and Identification of Main Compounds by LC/ESI-MS Analysis. International Journal of Food Science, 2019, 3721828.

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Antioxidant and Anti-inflammatory Assays

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Lipopolysaccharide (LPS) from sulforaphane, protein tyrosine phosphatase (PTP1B), rat lens aldose reductase (RLAR), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), peroxynitrite (ONOO⁻), Griess reagent, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), ethylenediaminetetraacetic acid (EDTA), 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride (AMT), p-nitrophenyl phosphate (pNPP), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), fetal bovine serum (FBS), and antibiotics were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from HyClone (Logan, UT, USA). Dihydrorhodamine 123 (DHR 123) was purchased from Molecular Probes (Eugene, OR, USA). Various primary antibodies (iNOS, COX-2, and β-actin) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Polyvinylidene fluoride (PVDF) membrane (Immobilon-P) was obtained from Millipore Co. (Billerica, MA, USA). SuperSignal^® West Pico Chemi-luminescent Substrate was obtained from Pierce Biotechnology, Inc. (Rockford, IL, USA). All other chemicals and solvents were purchased from E. Merck Fluka or Sigma-Aldrich unless stated otherwise.

Jung H.A., Karki S., Ehom N.Y., Yoon M.H., Kim E.J, & Choi J.S. (2014). Anti-Diabetic and Anti-Inflammatory Effects of Green and Red Kohlrabi Cultivars (Brassica oleracea var. gongylodes). Preventive Nutrition and Food Science, 19(4), 281-290.

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Antioxidant Capacity Evaluation Protocol

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2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, DPPH, 2,3,5-triphenyltetrazolium chloride (TPTZ), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 3-nonanone as internal standard (IS), and ferrozine were purchased from Sigma Chemical Co. (St. Louis, MO, United States). Sorbitol, glucose, fructose, sucrose, formic acid, acetic acid, lactic acid, malic acid, citric acid, gallic acid (GA), protocatechualdehyde (PRO), (+)-catechin (CAT), chlorogenic acid (CHL), caffeic acid (CAF), syringic acid (SYR), epicatechin (EPI), p-coumaric acid (p-CUM), rutin (RUT), phlorizin (PRI), quercetin (QUE), and phloretin (PRE) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). All other reagents used were of analytical grade.

Zhu Z., Zhang Y., Wang W., Sun S., Wang J., Li X., Dai F, & Jiang Y. (2022). Changes in Physicochemical Properties, Volatile Profiles, and Antioxidant Activities of Black Apple During High-Temperature Fermentation Processing. Frontiers in Nutrition, 8, 794231.

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