4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole

4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) is a fluorescent reagent for the sensitive and specific detection of thiols (Ex/Em: 380/510 nm). 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole has low fluorescence background, and good stability of fluorophor^[1]. In Vitro:4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole, as a highly reactive fluorescent derivatizing reagent, can undergo a nucleophilic substitution reaction with thiols (-SH) under mild alkaline conditions to generate a strongly fluorescent ABD-thiol adduct. This adduct can be manually determined by a fluorometer or detected with high sensitivity and selectivity after separation by reversed-phase high-performance liquid chromatography (HPLC)^[1].

Protocol (The following is our recommended protocol. This protocol is only a guideline and should be modified according to your specific needs).
1. Preparation of Reagents
ABD-F Preparation: Dissolve 1 mM of this product (ABD-F) in 0.1 M borate buffer (pH 8.0, Na⁺).
Standard/Sample: Solution of the thiol to be tested (such as cysteine, glutathione, etc.), dissolved in 0.1 M borate buffer (pH 8.0, Na⁺).
Protective agent: Disodium ethylenediaminetetraacetate (EDTA·2Na) (HY-Y0682A), added to the reaction buffer to a final concentration of 1-2 mM to chelate metal ions and prevent thiol oxidation.
Termination/Acidification reagent: 0.1 M hydrochloric acid (HCl).
2. Experimental Procedure
2.1 In a 5 mL glass reaction tube, add sequentially: 1 mL of 1 mM ABD-F solution (containing 1 mM EDTA) and 1.0 mL of thiol-containing sample/standard solution (containing 2 mM EDTA).
Note: To ensure complete reaction, the molar amount of ABD-F should be significantly excess over the thiol (refer to a 100-fold excess). For example, if the thiol concentration is 5 μM, then the ABD-F concentration should be 500 μM.
2.2 Vortex mix immediately and tighten the cap.
2.3 Place the reaction tube in a 50°C water bath or incubator and react in the dark for 5 minutes.
2.4 After the reaction is complete, immediately place the reaction tube in an ice-water bath to cool and terminate the reaction.
3. Fluorescence Measurement (using a fluorescence spectrophotometer as an example)
3.1 Add 0.6 mL of 0.1 M HCl to the cooled reaction solution and mix well. The final pH of the solution is approximately 2 at this point.
3.2 Measure using a fluorescence spectrophotometer. Reference parameter settings: Ex/Em: 380/510 nm. Note: A thiol-free ABD-F solution, processed using the same steps, serves as a reagent blank and is subtracted from the sample fluorescence value.