Laboratório de Imunopatologia Keizo Asami - LIKA, Universidade Federal de Pernambuco - UFPE, Av. Prof. Moraes Rego 1235, Recife, PE, 50670-901, Brazil.
Anal Bioanal Chem. 2019 Oct;411(25):6667-6676. doi: 10.1007/s00216-019-02040-5. Epub 2019 Aug 5.
Human epidermal growth factor receptor-2 (HER2) is an important biomarker in the diagnosis and prognosis of breast cancer. This work aimed to develop an aptasensor to detect HER2 in human serum. HER2 aptamer was immobilized by electrostatic adsorption on the surface of a homemade screen-printed electrode modified with poly-L-lysine. Measurements were made by differential pulse voltammetry using methylene blue as a redox indicator. A calibration curve was constructed (R = 0.997) using different concentrations of HER2 protein (10-60 ng/mL) in PBS buffer (pH 7.4), with a detection limit of 3.0 ng/mL. The aptasensor showed good reproducibility with relative standard deviations (RSDs) of 3% and remained stable for 3 days with an RSD around 2%. When the tests were performed with serum from a healthy woman, a peak of 6.72 μA was found without the addition of the protein. When we tested the serum of a woman with HER2+ breast cancer, we obtained a signal of 2.65 μA; the same pattern was found when adding to protein in serum control, i.e., the higher the concentration of protein, the lower the signal. The aptasensor was characterized by scanning electron microscopy and isothermal titration calorimetry (ITC), showing excellent interaction between aptamer and target protein. The results revealed a promising and sensitive tool capable of detecting HER2 protein in human serum with albumin depletion, aiding in the molecular diagnosis of breast cancer. Graphical abstract.
人表皮生长因子受体 2(HER2)是乳腺癌诊断和预后的重要生物标志物。本工作旨在开发一种适体传感器用于检测人血清中的 HER2。通过静电吸附将 HER2 适体固定在经过聚-L-赖氨酸修饰的自制丝网印刷电极表面。通过使用亚甲基蓝作为氧化还原指示剂的差分脉冲伏安法进行测量。使用不同浓度的 HER2 蛋白(10-60ng/mL)在 PBS 缓冲液(pH7.4)中构建校准曲线(R=0.997),检测限为 3.0ng/mL。该适体传感器具有良好的重现性,相对标准偏差(RSD)为 3%,在 3 天内保持稳定,RSD 约为 2%。当用健康女性的血清进行测试时,在不添加蛋白质的情况下发现了 6.72μA 的峰值。当我们测试 HER2+乳腺癌女性的血清时,我们得到了 2.65μA 的信号;在血清对照中添加蛋白质时也发现了相同的模式,即蛋白质浓度越高,信号越低。该适体传感器通过扫描电子显微镜和等温滴定量热法(ITC)进行了表征,显示了适体与靶蛋白之间的优异相互作用。结果表明,该适体传感器是一种有前途的灵敏工具,能够在白蛋白耗尽的情况下检测人血清中的 HER2 蛋白,有助于乳腺癌的分子诊断。