Proteases are involved in cancer' taking part in immune (dis)regulation, malignant progression and tumour growth. Recently, it has been found that expression levels of one of the members of the serine protease family, trypsin, is upregulated in human cancer cells of several organs, being considered as a specific cancer biomarker. Considering the great attention that electrochemical peptide sensors have nowadays, in this work, we propose a novel electroanalytical strategy for the determination of this important biomolecule. It implies the immobilization of a short synthetic peptide sequence, dually labelled with fluorescein isothiocyanate (FITC) and biotin, onto neutravidin-modified magnetic beads (MBs), followed by the peptide digestion with trypsin. Upon peptide disruption, the modified MBs were incubated with a specific fluorescein Fab fragment antibody labelled with horseradish peroxidase (HRP-antiFITC) and magnetically captured on the surface of a screen-printed carbon electrode (SPCE), where amperometric detection was performed using the hydroquinone (HQ)/HRP/H2O2 system. The biosensor exhibited a good reproducibility of the measurements (RSD 3.4%, n = 10), and specificity against other proteins and proteases commonly found in biological samples. This work reports the first quantitative data so far on trypsin expression in human cell lysates. The developed bioplatform was used for the direct determination of this protease in lysates from pancreatic cancer, cervix carcinoma and kidney cells in only 3 h and 30 min using low amounts (~ 0.1 μg) of raw extracts. Graphical abstract.

中文翻译：

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一种用于测定胰腺癌细胞中转移相关蛋白酶的新型肽基电化学生物传感器。

蛋白酶参与癌症的免疫（失调）调节、恶性进展和肿瘤生长。最近，已经发现丝氨酸蛋白酶家族的一个成员胰蛋白酶的表达水平在几个器官的人类癌细胞中上调，被认为是一种特定的癌症生物标志物。考虑到电化学肽传感器如今备受关注，在这项工作中，我们提出了一种新的电分析策略来确定这一重要的生物分子。这意味着将用异硫氰酸荧光素 (FITC) 和生物素双重标记的短合成肽序列固定在中性抗生物素蛋白修饰的磁珠 (MB) 上，然后用胰蛋白酶消化肽。在肽中断后，修饰后的 MB 与用辣根过氧化物酶 (HRP-antiFITC) 标记的特定荧光素 Fab 片段抗体一起孵育，并被磁性捕获在丝网印刷碳电极 (SPCE) 的表面上，其中使用对苯二酚 (HQ)/ HRP/H2O2 系统。该生物传感器表现出良好的测量重现性（RSD 3.4%，n = 10），以及对生物样品中常见的其他蛋白质和蛋白酶的特异性。这项工作报告了迄今为止关于人类细胞裂解物中胰蛋白酶表达的第一个定量数据。开发的生物平台用于使用少量（约 0.1 μg）的原始提取物在 3 小时和 30 分钟内直接测定胰腺癌、宫颈癌和肾细胞裂解物中的这种蛋白酶。图形概要。