A Fluorescent Probe for Diacetyl Detection

Abstract

A water-soluble fluorescent probe, rhodamine B hydrazide (RBH), was prepared and its properties for recognition of diacetyl were studied. The method employs the reaction of diacetyl with RBH, a colorless and non-fluorescent rhodamine B spiro form derivative to give a pink-colored fluorescent substance. In weakly acidic media, RBH reacts more selectively with diacetyl than with other carbonyls, causing a large increase in fluorescence intensity and thereby providing an easy assay for the determination of diacetyl.

Protein carbonyl determination by a rhodamine B hydrazide-based fluorometric assay

“…Fig. 2 ) is very similar to that obtained by the corresponding hydrazone formed by the reaction of RBH with diacetyl [20] . The maximum FSA increase of the protein carbonyl-RBH hydrazone that is caused by gndHCl may be due to its 3 amino groups compared to the 2 amino groups of the non-effective urea, and possibly because of its higher chaotropic potential than of urea [36] .…”

“…Rhodamine B hydrazide (RBH): RBH was synthesized according to a published procedure [20] , [22] . The product was recrystallized from methanol-acetone as a colorless solid with purity > 98% by TLC (yield: ~ 0.28 g), and identified by HPLC-MS. RBH is supplied by Synchem UG & Co. KG, Germany (order number: am005; http://www.synchem.de/chemical_Rhodamine%20B%20hydrazide.html ),…”

“…The following RBH assay procedure has been developed after standardization of its following parameters: assay reaction pH (previously studied for the reaction of RBH with diacetyl's carbonyls [20] ); assay reaction time, optimum pH and excitation/emission (ex/em) wavelengths for attaining maximum fluorescence (units, FU) of the protein carbonyl-RBH hydrazone, all in the presence of chaotropic factors (gndHCl and urea) in order to minimize reaction time and maximize hydrazone FU and stability over time; establishment of the molar stoichiometry of the reaction between RBH and protein carbonyl groups, which is 1:1 and allows the use of RBH for constructing the standard curve of the RBH assay. Standardization experiments were performed on certain control protein (BSA ox/red , lysozyme ox/red , pepsin ox/red ), and are presented in the Supplement's Part I.…”

“…1 ), which proceeds via the opening of the non-fluorescent cyclic spirolactam ring amide of RBH. This opening can take place either via a low pH-proton-induced conformation change of RBH [20] , or by the gndHCl in the assay buffer (33/17 mM C/P–8 M gndHCl, pH 5) as the stoichiometry 1:1 suggests, or during the reaction of RBH (at acidic pH) with the protein carbonyls (and the formation of the hydrazone bond). Therefore, the molar carbonyl content of a protein can be easily determined fluorometrically by the RBH assay, by using an RBH standard curve the slope of which sets the sensitivity limit of the RBH assay.…”

“…RBH has never been used for the quantification of protein carbonyls, except as a tag in reactive carbonyl containing short peptides (fused to T4 lysozyme or synthesized on filter paper for colorimetric assays of the peptide-hydrazide interaction), used for site-specific protein labeling [19] . The only time RBH has been used to quantify a hydrazone derivative was with diacetyl (by formation of a hydrazone bond with one of its two carbonyl groups [20] ); also for the quantification of malondialdehyde in biological fluids (via derivatization of its aldehyde group, and HPLC quantification [21] ). Other than these cases, RBH has been used for the quantification of the following molecules via their direct/indirect oxidation and decompose of RBH to its fluorescent product rhodamine B: peroxynitrite (via direct oxidation of RBH [22] ; H 2 O 2 (via iron-tetrasulfonatophthalocyanine catalysis), and also biomolecules that produce H 2 O 2 via catalysis by their oxidase (such as glucose via glucose oxidase) [23] ; Cu 2+ (by its binding to the spirolactam amide ring of RBH, thereby hydrolyzing it to rhodamine B [24] ); NO indirectly (it converts NO 2 aerobically to N 2 O 3 , which, via nitrosylation, converts the hydrazide amino group of RBH to diazonium group, the 5-membered ring of which is opened to form an azide intermediate that is converted into rhodamine B) [25] ; hemoglobin and cytochrome c (via RBH hydrolysis to rhodamine B after RBH incorporation in detergent sodium dodecylbenzene sulfonate micelles [26] , [27] ).…”