Real-time monitoring of distinct binding kinetics of hot-spot mutant p53 protein in human cancer cells using an individual nanorod-based plasmonic biosensor

• Published in: Sensors and actuators. B, Chemical Chemical, 2020-11, Vol.322, p.128584, Article 128584
• Main Authors: Song, Sojin, Lee, Jong Uk, Kang, Jinho, Park, Kyong Hwa, Sim, Sang Jun
• Format: Article
• Language: English
• Subjects: Biosensors; Breast cancer; Cancer; Gold nanoparticles (AuNPs); Human performance; Liquid chromatography; Mass spectrometry; Monitoring; Nanorods; p53 protein; Performance evaluation; Plasmonic biosensors; Plasmonics; Pretreatment; Proteins; Rayleigh scattering; Real time; Sensors
• ISSN: 0925-4005; 1873-3077
• DOI: 10.1016/j.snb.2020.128584

•Our nanoplasmonic biosensor has been developed to detect hot-spot mutant p53 protein utilizing the binding of the GADD45 promoter and protein.•Kinetic parameters of DNA-protein interactions were characterized to estimate equilibrium dissociation and relative transcriptional activities of wild-type and mutated p53 proteins.•Application of the kinetic parameters on the sensor for real-time kinetic analysis allowed highly accurate distinction of mutant protein with improved analytical performance.•Our nanoplasmonic biosensor is confirmed to have a satisfactory sensing performance for the application in clinical assay by comparing it with conventional analytical methods. Hot-spot mutant p53 proteins are highly associated with cancer malignancy and chemotherapy resistance of various cancers. Precise detection of the hot-spot mutant protein is therefore important in predicting therapeutic effects in cancer. However, conventional analytical methods such as liquid chromatography couple with tandem mass spectrometry (LC–MS/MS) and immunohistochemistry (IHC) have limited sensitivity as well as accuracy and require labor-intensive pretreatment steps. Here, an efficient individual nanorod-based plasmonic biosensor has been developed to detect hot-spot mutant p53 protein with the growth arrest and DNA damage 45 (GADD45) promoter. DNA-protein interactions of wild-type/mutant p53 proteins are kinetically analyzed on the biosensor surface through real-time monitoring of the localized surface plasmon resonance shift, and their dissociation constants and relative transcriptional activities are estimated. Hot-spot mutant proteins exhibited 14.29-fold higher in the dissociation constants and 19.91-fold lower in the relative transcriptional activities as compared to the wild-type proteins. These distinct kinetic values of hot-spot mutants allowed precise detection of hot-spot mutant protein and identification of mutated site of the protein using very small volume of clinical samples. We also demonstrated the clinical validation of the sensor by evaluating its performance in human breast tumor surgical specimens. The sensor’s ability to identify the mutant protein with high specificity and extremely low detection limit (11.47 fM) in comparison with the LC–MS/MS and IHC. The results confirmed that our sensor could be characterized as an efficient biosensor for application in clinical assays in terms of analytical performance.