title: Ultrasensitive electrochemical immunosensor based on dual signal amplification process for p16(INK4a) cervical cancer detection in clinical samples.
abstract: The p16(INK4a) (p16) is a cyclin-dependent kinase inhibitor, which has been evaluated in several studies as a diagnostic marker of cervical cancer. Immunostaining using p16 specific antibody has confirmed an over-expression of p16 protein in cervical cancer cells and its association with disease progression. This article reports an ultrasensitive electrochemical immunosensor for specific detection of p16 and demonstrates its performance for detection of solubilized p16 protein in cell lysates obtained from patients. Sandwich-based immunoreaction couple with double signal amplification strategy based on catalytic enlargement of particle tag was used for high sensitivity and specificity. The conditions were optimized to create an immunoassay protocol. Disposable screen-printed electrode modified with capture antibodies (Ab1) was selected for further implementation towards point-of-care diagnostics. Small gold nanoparticles (15 nm diameter) conjugated with detection antibodies (Ab2) were found to better serve as a detection label due to limited interference with antigen-antibody interaction. Double signal enhancement was performed by sequential depositions of gold and silver layers. This gave the sensitivity of 1.78 μA mL(ng GST-p16)(-1) cm(-2) and detection limit of 1.3 ng mL(-1) for GST-p16 protein which is equivalent to 0.49 ng mL(-1) for p16 protein and 28 cells for HeLa cervical cancer cells. In addition to purified protein, the proposed immunosensor effectively detected elevated p16 level in cervical swab samples obtained from 10 patients with positive result from standard Pap smear test, indicating that an electrochemical immunosensors hold an excellent promise for detection of cervical cancer in clinical setting.
authors.affiliation: Nanostructures and Functional Assembly Laboratory, National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand.
authors.forename: Pattasuda
authors.initials: P
authors.lastname: Duangkaew
authors.affiliation: Nanomolecular Target Discovery Laboratory, National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand.
authors.forename: Satita
authors.initials: S
authors.lastname: Tapaneeyakorn
authors.affiliation: Nanomolecular Target Discovery Laboratory, National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand.
authors.forename: Chayachon
authors.initials: C
authors.lastname: Apiwat
authors.affiliation: Nanomolecular Target Discovery Laboratory, National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand; Department of Immunology and Department of Obstetrics and Gynaecology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Tararaj
authors.initials: T
authors.lastname: Dharakul
authors.affiliation: Department of Immunology and Department of Obstetrics and Gynaecology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Somsak
authors.initials: S
authors.lastname: Laiwejpithaya
authors.affiliation: Trace Analysis and Biosensor Research Center, Department of Chemistry, Faculty of Science, Prince of Songkla University, Songkhla, Thailand.
authors.forename: Proespichaya
authors.initials: P
authors.lastname: Kanatharana
authors.affiliation: Nanostructures and Functional Assembly Laboratory, National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand. Electronic address: rawiwan.lao@nanotec.or.th.
authors.forename: Rawiwan
authors.initials: R
authors.lastname: Laocharoensuk
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis*; D015374:Biosensing Techniques / Q000295:instrumentation; D003217:Conductometry / Q000295:instrumentation*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000032:analysis*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018511:Systems Integration; D002583:Uterine Cervical Neoplasms / Q000737:chemistry* / Q000175:diagnosis*
chemical_list: D014408:Biomarkers, Tumor; D019941:Cyclin-Dependent Kinase Inhibitor p16
keywords: Cancer diagnostics; Cervical cancer detection; Electrochemical immunosensor