title: Label-free fluorescence detection of kinase activity using a gold nanoparticle based indicator displacement assay.
abstract: A straightforward indicator-displacement assay (IDA) has been developed for the quantitative analysis of ATP→ADP conversion. The IDA relies on the use of gold nanoparticles passivated with a monolayer of thiols terminating with a 1,4,7-triazacyclononane (TACN)·Zn(2+) head group. The analytes ATP and ADP compete to a different extent with a fluorescent probe for binding to the monolayer surface. In the presence of ATP the fluorescent probe is free in solution, whereas in the presence of ADP the fluorescent probe is captured by the nanoparticles and its fluorescence is quenched. The linear response of the fluorescence signal towards different ratios of ATP : ADP permitted the detection of protein kinase activity simply by adding aliquots of the enzyme solution to the assay solution followed by measurement of the fluorescent intensity. The assay poses no restrictions on the target kinase nor does it require labeling of the kinase substrate. The assay was tested on the protein kinases PIM-1 and Src and validated through a direct comparison with the classical radiometric assay using the [γ-(32)P]-labeled ATP.
authors.affiliation: Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131 Padova, Italy. leonard.prins@unipd.it.
authors.forename: Cristian
authors.initials: C
authors.lastname: Pezzato
authors.forename: Davide
authors.initials: D
authors.lastname: Zaramella
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Martinelli
authors.forename: Grégory
authors.initials: G
authors.lastname: Pieters
authors.forename: Mario A
authors.initials: MA
authors.lastname: Pagano
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Prins
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D051573:Proto-Oncogene Proteins c-pim-1 / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D000244:Adenosine Diphosphate; D006046:Gold; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D019061:src-Family Kinases; D051573:Proto-Oncogene Proteins c-pim-1