Mucelli S Pozzi, Zamuner M, Tormen M, Stanta G, Ugo P
Molecular Histopathology Laboratory, ICGEB, Padriciano 99, 34012 Trieste, Italy.
Biosens Bioelectron. 2008 Jul 15;23(12):1900-3. doi: 10.1016/j.bios.2008.02.027. Epub 2008 Mar 8.
In this study we demonstrate the possibility to prepare highly sensitive nanostructured electrochemical immunosensors by immobilizing biorecognition elements on nanoelectrode ensembles (NEEs) prepared in track-etch polycarbonate membranes. The gold nanodisk electrodes act as electrochemical transducers while the surrounding polycarbonate binds the antibody-based biorecognition layer. The interaction between target protein and antibody is detected by suitable secondary antibodies labelled with a redox enzyme. A redox mediator, added to the sample solution, shuttles electrons from the nanoelectrodes to the biorecognition layer, so generating an electrocatalytic signal. This allows one to fully exploit the highly improved signal-to-background current ratio, typical of NEEs. In particular, the receptor protein HER2 was studied as the target analyte. HER2 detection allows the identification of breast cancer that can be treated with the monoclonal antibody trastuzumab. NEEs were functionalized with trastuzumab which interacts specifically with HER2. The biorecognition process was completed by adding a primary antibody and a secondary antibody labelled with horseradish peroxidase. Hydrogen peroxide was added to modulate the label electroactivity; methylene blue was the redox mediator generating voltammetric signals. NEEs functionalized with trastuzumab were tested to detect small amounts of HER2 in diluted cell lysates and tumour lysates.
在本研究中，我们展示了通过将生物识别元件固定在径迹蚀刻聚碳酸酯膜中制备的纳米电极阵列（NEEs）上，来制备高灵敏度纳米结构电化学免疫传感器的可能性。金纳米盘电极作为电化学换能器，而周围的聚碳酸酯则结合基于抗体的生物识别层。靶蛋白与抗体之间的相互作用通过用氧化还原酶标记的合适二抗进行检测。添加到样品溶液中的氧化还原介质将电子从纳米电极穿梭到生物识别层，从而产生电催化信号。这使得人们能够充分利用NEEs典型的显著提高的信号与背景电流比。特别地，将受体蛋白HER2作为靶分析物进行了研究。HER2检测能够识别可用单克隆抗体曲妥珠单抗治疗的乳腺癌。NEEs用与HER2特异性相互作用的曲妥珠单抗进行功能化。通过添加一抗和用辣根过氧化物酶标记的二抗来完成生物识别过程。添加过氧化氢以调节标记物的电活性；亚甲蓝是产生伏安信号的氧化还原介质。对用曲妥珠单抗功能化的NEEs进行了测试，以检测稀释的细胞裂解物和肿瘤裂解物中的少量HER2。
