![Gold nanoparticle-based electrochemical detection of protein phosphorylation [An article from: Analytica Chimica Acta]](https://www.ebooknetworking.net/books/B00/0PD/bigB000PDYI6Y.jpg)
Gold nanoparticle-based electrochemical detection of protein phosphorylation [An article from: Analytica Chimica Acta]
Description
This digital document is a journal article from Analytica Chimica Acta, published by Elsevier in 2007. The article is delivered in HTML format and is available in your Amazon.com Media Library immediately after purchase. You can view it with any web browser.
Description:
In this report, we demonstrate the application of Au nanoparticles in the electrochemical detection of protein phosphorylation. The method is based on the labeling of a specific phosphorylation event with Au nanoparticles, followed by electrochemical detection. The phosphorylation reaction is coupled with the biotinylation of the kinase substrate using a biotin-modified adenosine 5'-triphosphate [@c]-biotinyl-3,6,9-trioxaundecanediamine (ATP) as the co-substrate. When the phosphorylated and biotinylated kinase substrate is exposed to streptavidin-coated Au nanoparticles, the high affinity between the streptavidin and biotin resulted in the attachment of Au nanoparticles on the kinase substrate. The electrochemical response obtained from Au nanoparticles enables monitoring the activity of the kinase and its substrate, as well as the inhibition of small molecule inhibitors on protein phosphorylation. We determined the activity of Src non-receptor protein tyrosine kinase, p60^c^-^S^r^c and protein kinase A in combination with their highly specific substrate peptides Raytide(TM) EL and Kemptide, respectively. The detection limits for Raytide(TM) EL and Kemptide were determined as 5 and 10@mM, (S/N=3), and the detection limits for the kinase activity of p60^c^-^S^r^c and protein kinase A (PKA) were determined as 5 and 10UmL^-^1, (S/N=3), respectively. Tyrosine kinase reactions were also performed in the presence of a well-defined inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2), and its negative control molecule, 4-amino-7-phenylpyrazol[3,4-d] pyrimidine (PP3), which had no inhibition effect. Based on the dependency of Au nanoparticle signal on inhibitor concentration, IC"5"0 value, half-maximal inhibition of the inhibitors was estimated. IC"5"0 values of PP2, genistein and herbimycin A to p60^c^-^S^r^c were detected as 5nM, 25@mM and 900nM, respectively. The inhibition of PKA activity on Kemptide using ellagic acid was monitored with an IC"5"0 of 3.5@mM. The performance of the biosensor was optimized including the kinase reaction, incubation with streptavidin-coated Au nanoparticles, and the small molecule inhibitors. Kinase peptide-modified electrochemical biosensors are promising candidates for cost-effective kinase activity and inhibitor screening assays.
Description:
In this report, we demonstrate the application of Au nanoparticles in the electrochemical detection of protein phosphorylation. The method is based on the labeling of a specific phosphorylation event with Au nanoparticles, followed by electrochemical detection. The phosphorylation reaction is coupled with the biotinylation of the kinase substrate using a biotin-modified adenosine 5'-triphosphate [@c]-biotinyl-3,6,9-trioxaundecanediamine (ATP) as the co-substrate. When the phosphorylated and biotinylated kinase substrate is exposed to streptavidin-coated Au nanoparticles, the high affinity between the streptavidin and biotin resulted in the attachment of Au nanoparticles on the kinase substrate. The electrochemical response obtained from Au nanoparticles enables monitoring the activity of the kinase and its substrate, as well as the inhibition of small molecule inhibitors on protein phosphorylation. We determined the activity of Src non-receptor protein tyrosine kinase, p60^c^-^S^r^c and protein kinase A in combination with their highly specific substrate peptides Raytide(TM) EL and Kemptide, respectively. The detection limits for Raytide(TM) EL and Kemptide were determined as 5 and 10@mM, (S/N=3), and the detection limits for the kinase activity of p60^c^-^S^r^c and protein kinase A (PKA) were determined as 5 and 10UmL^-^1, (S/N=3), respectively. Tyrosine kinase reactions were also performed in the presence of a well-defined inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2), and its negative control molecule, 4-amino-7-phenylpyrazol[3,4-d] pyrimidine (PP3), which had no inhibition effect. Based on the dependency of Au nanoparticle signal on inhibitor concentration, IC"5"0 value, half-maximal inhibition of the inhibitors was estimated. IC"5"0 values of PP2, genistein and herbimycin A to p60^c^-^S^r^c were detected as 5nM, 25@mM and 900nM, respectively. The inhibition of PKA activity on Kemptide using ellagic acid was monitored with an IC"5"0 of 3.5@mM. The performance of the biosensor was optimized including the kinase reaction, incubation with streptavidin-coated Au nanoparticles, and the small molecule inhibitors. Kinase peptide-modified electrochemical biosensors are promising candidates for cost-effective kinase activity and inhibitor screening assays.
