title: Electrochemical biosensor for protein kinase A activity assay based on gold nanoparticles-carbon nanospheres, phos-tag-biotin and β-galactosidase.
abstract: A sensitive and selective electrochemical biosensor was fabricated for protein kinase A (PKA) activity assay. Multiple signal amplification techniques were employed including the nanocomposite of gold nanoparticles and carbon nanospheres (Au@C), the biocomposite of SiO2 and streptavidin (SiO2-SA), the composite of AuNPs and biotinylated β-galactosidase (AuNPs-B-Gal) and in situ enzymatic generation of electrochemical activity molecule of p-aminophenol. After peptides were assembled on Au@C modified electrode surface, they were phosphorylated by PKA in the presence of ATP. Then, biotinylated Phos-tag was modified on electrode surface through the specific interaction between Phos-tag and phosphate group. Finally, SiO2-SA and AuNPs-B-Gal were captured through the specific interaction between biotin and streptavidin. Because the electrochemical response of p-aminophenol was directly related to PKA concentration, an innovative electrochemical assay could be realized for PKA detection. The detection limit was 0.014unit/mL. The developed method showed high detection sensitivity and selectivity. In addition, the fabricated biosensor can be also applied to detect PKA in human normal gastricepithelial cell line and human gastric carcinoma cell line with satisfactory results.
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, PR China; Department of Animal Science, Shandong Agricultural University, Taian 271018, PR China.
authors.forename: Yunlei
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, PR China. Electronic address: yinhs@sdau.edu.cn.
authors.forename: Huanshun
authors.initials: H
authors.lastname: Yin
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, PR China.
authors.forename: Xue
authors.initials: X
authors.lastname: Li
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, PR China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Li
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, PR China.
authors.forename: Shiyun
authors.initials: S
authors.lastname: Ai
authors.affiliation: Department of Animal Science, Shandong Agricultural University, Taian 271018, PR China. Electronic address: hailin@sdau.edu.cn.
authors.forename: Hai
authors.initials: H
authors.lastname: Lin
mesh_terms: D000627:Aminophenols / Q000032:analysis / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D019921:Biotinylation; D002244:Carbon / Q000737:chemistry*; D002460:Cell Line; D045744:Cell Line, Tumor; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000378:metabolism; D055664:Electrochemical Techniques / Q000379:methods; D006046:Gold / Q000737:chemistry*; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry*; D054118:Nanospheres / Q000737:chemistry*; D019809:Streptavidin / Q000737:chemistry; D001616:beta-Galactosidase / Q000737:chemistry*
chemical_list: D000627:Aminophenols; D002244:Carbon; D006046:Gold; D019809:Streptavidin; D017868:Cyclic AMP-Dependent Protein Kinases; D001616:beta-Galactosidase; C026729:4-aminophenol
keywords: Biotinylated Phos-tag; Biotinylated β-galactosidase; Electrochemical biosensor; Multiple signal amplification; Protein kinase A