Determination of phosphate in nanomolar range by an enzyme-coupling fluorescent method.
We have successfully configured a new ultrasensitive fluorescent phosphate assay that detects free phosphate in solution through the formation of the fluorescent product resorufin. The phosphate assay relies on coupling phosphate generation to purine nucleoside phosphorylase, xanthine oxidase, and horseradish peroxidase. The response is excellent in the nanomolar range, being linear between 50 nM and 5 microM phosphate. This method is more sensitive (more than 10-fold) than other reported methods and is amenable to miniaturization. In particular, we have demonstrated the utility of this new method in a format suitable for ultra-high-throughput screening.
Vazquez
Maria J
MJ
GlaxoSmithKline Pharmaceuticals, Centro de Investigación Básica, Santiago Grisolía 4, Tres Cantos 28760, Madrid, Spain. Maria-Jesus.Vazquez@gsk.com
Rodriguez
Beatriz
B
Zapatero
Cleofe
C
Tew
David G
DG
D019883:Alkyl and Aryl Transferases / Q000032:analysis / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D005453:Fluorescence; D006735:Horseradish Peroxidase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010078:Oxazines / Q000378:metabolism; D010710:Phosphates / Q000032:analysis* / Q000378:metabolism; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism; D013997:Time Factors; D014969:Xanthine Oxidase / Q000378:metabolism
D010078:Oxazines; D010710:Phosphates; C014180:resorufin; D006735:Horseradish Peroxidase; D014969:Xanthine Oxidase; D011683:Purine-Nucleoside Phosphorylase; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase