Novel type of general protein assay using a chromogenic and fluorogenic amine-reactive probe.
We report on a new and simple one-reagent method for general protein assay. It makes use of one of two new reactive labeling reagents presented here (and referred to as pyrylium [Py] labels). These can be applied for both photometric and fluorometric protein assays at near neutral pHs at room temperature. The Py labels undergo a large spectral change on conjugation to the amino group of proteins and typically change their color from blue to red. Therefore, and unlike in other assays, there is no need to separate the unconjugated (blue) label from the red conjugate, which can be determined by direct photometry with a limit of detection of 1.2 microg/ml for human serum albumin. The assay can be extended to fluorometry because the fluorescence of the free Py label is weak (with a quantum yield of <1%) but increases strongly (to >40%) on conjugation. The strong fluorescence of the red conjugates can be determined directly and without interference by the blue (and weakly fluorescent) free label. The fluorometric assay resulted in a limit of detection of 60 ng/ml for bovine serum albumin (BSA). Validation of the fluorescence assay of blood plasma samples spiked with BSA gave recoveries in the range from 91 to 103%.
Hoefelschweiger
Bianca K
BK
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany.
Duerkop
Axel
A
Wolfbeis
Otto S
OS
D000588:Amines / Q000737:chemistry; D002021:Buffers; D002138:Calibration; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry; D006575:Heterocyclic Compounds, 3-Ring / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010783:Photometry; D011506:Proteins / Q000032:analysis*; D012709:Serum Albumin / Q000032:analysis; D012710:Serum Albumin, Bovine / Q000032:analysis
D000588:Amines; D002021:Buffers; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D011506:Proteins; D012709:Serum Albumin; C505939:pyrylium 1; C505940:pyrylium 6; D012710:Serum Albumin, Bovine