Nanostructures and Functional Assembly Laboratory, National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand.
Nanomolecular Target Discovery Laboratory, National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand.
Biosens Bioelectron. 2015 Dec 15;74:673-9. doi: 10.1016/j.bios.2015.07.004. Epub 2015 Jul 13.
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.
p16(INK4a) (p16) 是一种细胞周期蛋白依赖性激酶抑制剂，已在多项研究中被评估为宫颈癌的诊断标志物。使用 p16 特异性抗体的免疫染色已证实宫颈癌细胞中 p16 蛋白的过度表达及其与疾病进展的相关性。本文报道了一种用于特异性检测 p16 的超灵敏电化学免疫传感器，并展示了其在检测来自患者的细胞裂解物中可溶性 p16 蛋白方面的性能。基于夹心的免疫反应与基于粒子标记物催化放大的双重信号放大策略相结合，用于实现高灵敏度和特异性。优化了条件以创建免疫分析协议。选择带有捕获抗体 (Ab1) 的一次性丝网印刷电极进行进一步实施，以实现即时护理诊断。与检测抗体 (Ab2) 偶联的小 15nm 直径的金纳米粒子由于与抗原-抗体相互作用的干扰有限，被发现更适合作为检测标签。通过顺序沉积金和银层来实现双重信号增强。这使得 GST-p16 蛋白的灵敏度为 1.78 μA mL(ng GST-p16)(-1) cm(-2)，检测限为 1.3 ng mL(-1)，相当于 p16 蛋白的 0.49 ng mL(-1)和 HeLa 宫颈癌细胞的 28 个细胞。除了纯化蛋白外，该免疫传感器还能有效检测来自 10 名巴氏涂片检查阳性的患者宫颈拭子样本中升高的 p16 水平，这表明电化学免疫传感器在临床环境中具有出色的宫颈癌检测潜力。
