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| Targets |
The primary target of o-Dianisidine dihydrochloride is peroxidase, an enzyme that catalyzes the oxidation of substrates in the presence of hydrogen peroxide. The compound serves as a chromogenic substrate for peroxidase, producing a colored product that can be detected spectrophotometrically. In ELISA and other immunoassays, peroxidase is conjugated to antibodies or other detection molecules, and the substrate is used to generate a signal that is proportional to the amount of target analyte. The compound's sensitivity and stability make it a valuable tool for colorimetric detection in biochemical assays.
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| ln Vitro |
In vitro, o-Dianisidine dihydrochloride is used as a colorimetric substrate for peroxidase in various biochemical assays. The compound is oxidized by peroxidase in the presence of hydrogen peroxide, producing an orange-colored soluble end product that can be measured spectrophotometrically at a suitable wavelength (typically around 450 nm). The intensity of the color is proportional to the peroxidase activity, which in turn is proportional to the amount of target analyte in the sample. The substrate is used in ELISA for the detection of antigens, antibodies, and other biomolecules. It is also used for the semi-quantitative measurement of lactate, uric acid, and other analytes.
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| ln Vivo |
In vivo studies of o-Dianisidine dihydrochloride are not applicable, as the compound is used as an in vitro research reagent and is not intended for in vivo use. The compound is not administered to animals or humans. It is used exclusively in biochemical and diagnostic assays. No extensive in vivo pharmacological or toxicological studies have been reported.
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| Enzyme Assay |
For peroxidase activity assays, o-Dianisidine dihydrochloride is dissolved in an appropriate buffer (e.g., 50 mM citrate-phosphate buffer, pH 5.0). The substrate is typically used at concentrations ranging from 0.1 to 1 mg/mL. Hydrogen peroxide is added to the reaction mixture to initiate the reaction. The reaction is incubated at room temperature or 37°C for a specified time, and the color development is monitored by measuring absorbance at a suitable wavelength (e.g., 450 nm). The reaction can be stopped by the addition of a stop solution (e.g., sulfuric acid), and the absorbance is measured. The amount of product formed is proportional to the peroxidase activity. For ELISA applications, the substrate is added to wells containing peroxidase-conjugated detection antibodies, and the color development is measured.
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| Cell Assay |
For cellular studies, o-Dianisidine dihydrochloride is typically not used in cell-based assays, as it is a substrate for enzymatic assays. However, it may be used to measure peroxidase activity in cell lysates or supernatants. Cells are cultured and lysed, and the lysate is incubated with the substrate and hydrogen peroxide. The color development is measured spectrophotometrically. The substrate is also used in immunohistochemistry and other staining applications.
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| Animal Protocol |
Standard in vivo protocols for o-Dianisidine dihydrochloride are not applicable, as the compound is used as an in vitro research reagent. It is not administered to animals. No specific in vivo protocols are reported in the literature.
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| ADME/Pharmacokinetics |
Pharmacokinetic data for o-Dianisidine dihydrochloride are not applicable, as the compound is used as an in vitro research reagent and is not intended for in vivo use. The compound is not administered systemically, and no ADME studies have been reported.
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| Toxicity/Toxicokinetics |
Toxicological data for o-Dianisidine dihydrochloride are limited, as the compound is used as a research reagent. The compound is generally considered to have low toxicity at the concentrations used for enzymatic assays. However, as a benzidine derivative, it may have potential mutagenic properties. Appropriate safety precautions should be taken when handling o-Dianisidine dihydrochloride, including the use of personal protective equipment and work in a well-ventilated area. The compound should be handled according to standard laboratory safety protocols.
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| Additional Infomation |
According to the National Toxicology Program (NTP), 3,3'-dimethoxybenzidine dihydrochloride is carcinogenic. 3,3'-Dimethoxybenzidine dihydrochloride is a grayish-white powder. (NTP, 1992) It is a highly toxic compound that can cause skin irritation and allergic reactions. It is used in the production of azo dyes.
o-Dianisidine dihydrochloride (CAS 20325-40-0) is a colorimetric peroxidase substrate used in ELISA and other biochemical assays. It has a molecular formula of C₁₄H₁₆N₂O₂·2HCl and a molecular weight of 317.21. The compound produces an orange-colored soluble end product when oxidized by peroxidase in the presence of hydrogen peroxide. It is used to semi-quantitatively measure lactate, uric acid, and other analytes. o-Dianisidine dihydrochloride is a valuable tool for colorimetric detection in biochemical research and is strictly for research use only. |
| Exact Mass |
316.074
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| CAS # |
20325-40-0
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| Related CAS # |
119-90-4 (Parent)
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| PubChem CID |
62311
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| Appearance |
Pale purple to purple solid powder
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| Boiling Point |
391.3ºC at 760 mmHg
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| Melting Point |
268 °C
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| Flash Point |
206℃
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| LogP |
5.301
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
20
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| Complexity |
236
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
UXTIAFYTYOEQHV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C14H16N2O2.2ClH/c1-17-13-7-9(3-5-11(13)15)10-4-6-12(16)14(8-10)18-2;;/h3-8H,15-16H2,1-2H3;2*1H
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| Chemical Name |
4-(4-amino-3-methoxyphenyl)-2-methoxyaniline;dihydrochloride
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O : ~33.33 mg/mL (~105.07 mM)
DMSO : ~12.5 mg/mL (~39.41 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.25 mg/mL (3.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 1.25 mg/mL (3.94 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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