Chinnappan Raja, Al Attas Sana, Kaman Wendy E, Bikker Floris J, Zourob Mohammed
Department of Chemistry, Alfaisal University, Al Zahrawi Street, Al Maather, Al Takhassusi Rd, Riyadh 11533, Saudi Arabia.
Department of Biological Sciences, College of Science, King abdulAziz University, Jeddah, Saudi Arabia.
Anal Biochem. 2017 Apr 15;523:58-64. doi: 10.1016/j.ab.2017.02.009. Epub 2017 Feb 17.
Mastitis in dairy cattle is an inflammatory reaction of the udder tissue. Mastitis increases plasmin levels, leading to an increased proteolysis of milk proteins such as casein, resulting in a significant decrease in milk quality and related dairy products. Due to its key-role in mastitis, we used plasmin proteolytic activity as a biomarker for the detection of mastitis in bovine mastitic milk. Inspired by earlier studies on protease activity using mastitic milk samples, we developed a simple colorimetric assay to distinguish mastitic milk from milk derived from healthy animals. The plasmin substrate coupled to magnetic nanoparticles form a black self-assembled monolayer on a gold sensor surface. In the presence of increased levels of plasmin, the substrate is cleaved and the peptide fragment attached to the magnetic beads, will be attracted by the magnet which is present under the sensor strips revealing the golden surface. We found the area of the golden color surface proportional to plasmin activity. The sensitivity of this method was determined to be 1 ng/ml of plasmin in vitro. Next, we tested the biosensor using mastitis positive milk of which infection is confirmed by bacterial cultures. This newly developed colorimetric biosensor has high potential in applications for the diagnosis of mastitis with potential spin offs to health, food and environmental sectors.
奶牛乳腺炎是乳腺组织的一种炎症反应。乳腺炎会提高纤溶酶水平，导致酪蛋白等乳蛋白的蛋白水解增加，从而使牛奶质量及相关乳制品显著下降。由于纤溶酶在乳腺炎中起关键作用，我们将纤溶酶的蛋白水解活性用作检测患乳腺炎奶牛乳汁中乳腺炎的生物标志物。受早期使用患乳腺炎乳汁样本研究蛋白酶活性的启发，我们开发了一种简单的比色测定法，以区分患乳腺炎奶牛的乳汁与健康奶牛的乳汁。与磁性纳米颗粒偶联的纤溶酶底物在金传感器表面形成黑色自组装单分子层。在纤溶酶水平升高时，底物被切割，附着在磁珠上的肽片段会被传感器条下方的磁铁吸引，从而露出金色表面。我们发现金色表面的面积与纤溶酶活性成正比。该方法在体外的灵敏度测定为纤溶酶1纳克/毫升。接下来，我们使用经细菌培养确诊感染的乳腺炎阳性乳汁对生物传感器进行了测试。这种新开发的比色生物传感器在乳腺炎诊断应用中具有很高的潜力，可能会衍生到健康、食品和环境领域。
