Evaluation of a tertiary amine labeling protocol for peptides and proteins using Ru(bpy)(3)(3+)-based chemiluminescence detection
by Waguespack, Brent Lyman; Lillquist, Amy; Townley, Jonathan C.; Bobbitt II, Donald R.
The properties of four ruthenium complexes and their efficiency as probes for chemiluminescent (CL) detection were evaluated, and a protocol for CL detection of a protein labeled with a tertiary amine was proposed. Of the four ruthenium complexes studied, Ru(bpy)(3)(3+) proved to exhibit the characteristics necessary for optimum CL detection. Two biotin derivatives were synthesized containing a tertiary amine, functionality and then incubated with the protein, avidin. The addition of the tertiary amine moiety to biotin made the derivative more sensitive to Ru(bpy)(3)(3+)-based CL. Quantitative data were obtained as a model of a labeling protocol by varying the number of biotins per avidin. Limit of detection (LOD) for either derivative were determined to be similar to 50 nM, for the lowest concentration studied. The relative luminescence response increased with increasing number of tertiary amine labels per molecule of avidin. The results showed the potential of this approach for the detection of proteins using this tertiary amine labeling methodology. (C) 2001 Elsevier Science B.V. All rights reserved.
- Analytica Chimica Acta
- Start Page
- 1873-4324; 0003-2670