基于柔性电极和信号放大技术构建检测磷脂酶C的电化学传感器

Fabrication of an Electrochemical Sensor for Phospholipase C Detection Based on Flexible Electrodes and Signal Amplification Technology

磷脂酶C(PLC)是动植物中重要的脂质水解酶,在哺乳动物细胞的代谢、生长、炎症、分化、衰老和凋亡中起着重要作用,与许多癌症的发生、发展显著相关。蛋白质催化原子转移自由基聚合(PATRP)是高分子设计、制备的有效手段,可通过链式反应将数百个电活性单体连接成一个高相对分子质量的聚合物链,从而使分子识别信号大大增强。首先,在柔性碳布(CC)电极表面修饰纳米金(AuNPs),之后通过3-巯基丙酸、碳二亚胺盐酸盐和N-羟基丁二酰亚胺,将PLC的特异性底物磷脂酰乙醇胺(PE)固定在电极上,经过PLC对PE的特异性水解产生磷酸基团,引入Zr^(4+),通过Zr^(4+)的配位作用将聚合引发剂α-溴苯乙酸修饰到电极表面,最后在蛋白质催化作用下进行2,2,6,6-四甲基哌啶氧化物(TEMPO)的PATRP聚合反应,在电极表面引入电活性聚合物分子链,构建信号放大型电化学传感器P(TEMPO)/PE/AuNPs/CC,用于磷脂酶C的高灵敏度检测。结果表明,该传感器检测PLC的线性范围为10~1×10^(5)mU/L,检出限为3.568 mU/L(S/N=3)。与其他检测PLC的方法相比,该传感器具有较宽的检测范围和较低的检出限,且在检测实际乳腺癌细胞中的PLC方面有巨大的实际应用潜力。

Phospholipase C(PLC)is an important lipid hydrolase in plants and animals,which plays an important role in the metabolism,growth,inflammation,differentiation,senescence,and apoptosis of mammalian cells,and is significantly associated with the onset and progression of many cancers.Proteincatalyzed atom transfer radical polymerization(PATRP)is an effective method of designing and preparing macromolecules,and it can be used to link hundreds of monomers by a chain reaction to form a highmolecular-mass polymer,so that the molecular recognition signals are amplified hundreds of times.In this experiment,gold nanoparticles(Au NPs)were first deposited on the surface of flexible carbon cloth(CC)electrode,and then the PLC-specific substrate phosphatidylethanolamine(PE)was immobilized to the electrode by 3-mercaptopropionic acid,carbodiimide hydrochloride and n-hydroxysuccinimide.After the phosphoric acid group was produced by the specific hydrolysis of PE by PLC,and Zr^(4+)was introduced,the polymerization initiatorα-bromophenylacetic acid was modified to the electrode surface by the coordination of Zr^(4+).Finally,PATRP of 2,2,6,6-tetramethyl-1-piperidinyloxy(TEMPO)was carried out,and electroactive polymers chains were introduced to the electrode surface.Thus a signal amplification electrochemical sensor[P(TEMPO)/PE/Au NPs/CC]was fabricated to detect PLC with high sensitivity.The results show that the sensor detected PLC with a linear range of 10~1×10^(5)mU/L and a detection limit of 3.568 mU/L(S/N=3).Compared with other methods for the detection of PLC,the sensor has a wider detection range and lower detection limit,and it has a huge practical application potential in detecting PLC in real breast cancer cells.