Tuteja Satish K, Duffield Todd, Neethirajan Suresh
BioNano Laboratory, School of Engineering, University of Guelph, Guelph, ON N1G 2W1, Canada.
J Mater Chem B. 2017 Sep 7;5(33):6930-6940. doi: 10.1039/c7tb01382e. Epub 2017 Aug 11.
Elevated circulating concentrations of non-esterified fatty acids (NEFA) and beta hydroxy butyrate (βHBA) in biological fluids are recognized as critical biomarkers for early diagnosis of negative energy balance (NEB) in dairy cows. Herein, we report the development of a cost-effective, bio-friendly and electrochemically active dual screen printed electrode (SPE) sensor platform composed of electro-reduced graphene oxide nanosheets (E-rGO) modified with specific antibodies against NEFA and βHBA. The chemically synthesized graphene oxide (GO) was reduced directly on the screen printed electrode (SPE) surface via a green electrochemical approach without using toxic chemicals. The E-rGO was characterized using various analytical techniques, like XPS, SEM, TEM, AFM, UV-Vis, Raman spectroscopy, FTIR, and XRD, to get insight into its properties. Electrochemical analysis demonstrates that the E-rGO-modified SPE electrodes exhibit enhanced and durable redox properties as compared to the pristine graphite and GO electrodes. Target specificity is accomplished through immobilization of specific antibodies against NEFA and βHBA over the nanostructure-modified surface of the SPE, which only interacts with its counterpart NEFA and βHBA only. The antibodies retain their characteristic immuno-complex formation property upon immobilization and exhibit changes to amperometric signals upon interaction with various concentrations of NEFA and βHBA in standard, spiked blood and real clinical samples. The DPV signals resulted from the developed immunosensor platform exhibited a good correlation (R∼ 0.99 for both NEFA and βHBA) for a wide range of target concentrations from 0.1 mM to 10 mM. The proposed immunosensor design not only provides a rapid analytical response time (≥1 min), but also simplicity in fabrication and instrumentation, which may provide a promising approach for on-farm diagnostics of ketosis and metabolic disorders associated with NEB.
生物体液中循环的非酯化脂肪酸（NEFA）和β-羟基丁酸（βHBA）浓度升高被认为是奶牛负能量平衡（NEB）早期诊断的关键生物标志物。在此，我们报告了一种具有成本效益、生物友好且具有电化学活性的双丝网印刷电极（SPE）传感器平台的开发，该平台由用针对NEFA和βHBA的特异性抗体修饰的电还原氧化石墨烯纳米片（E-rGO）组成。通过绿色电化学方法在丝网印刷电极（SPE）表面直接还原化学合成的氧化石墨烯（GO），无需使用有毒化学物质。使用各种分析技术对E-rGO进行表征，如XPS、SEM、TEM、AFM、UV-Vis、拉曼光谱、FTIR和XRD，以深入了解其性质。电化学分析表明，与原始石墨和GO电极相比，E-rGO修饰的SPE电极表现出增强且持久的氧化还原特性。通过将针对NEFA和βHBA的特异性抗体固定在SPE的纳米结构修饰表面上来实现目标特异性，该表面仅与对应的NEFA和βHBA相互作用。抗体在固定后保留其特征性免疫复合物形成特性，并在与标准、加标血液和实际临床样品中不同浓度的NEFA和βHBA相互作用时表现出安培信号变化。所开发的免疫传感器平台产生的DPV信号在0.1 mM至10 mM的广泛目标浓度范围内表现出良好的相关性（NEFA和βHBA的R均约为0.99）。所提出的免疫传感器设计不仅提供了快速的分析响应时间（≥1分钟），而且制造和仪器简单，这可能为农场现场诊断酮病和与NEB相关的代谢紊乱提供一种有前景的方法。
