Brandenburg University of Technology Cottbus-Senftenberg, Universitätsplatz 1, 01968, Senftenberg, Germany.
Faculty of Mathematics and Informations Science, Warsaw University of Technology, plac Politechniki 1, 00-661, Warsaw, Poland.
Anal Bioanal Chem. 2019 Nov;411(29):7725-7735. doi: 10.1007/s00216-019-02199-x. Epub 2019 Nov 23.
The rapid and simultaneous detection of DNA and protein biomarkers is necessary to detect the outbreak of a disease or to monitor a disease. For example, cardiovascular diseases are a major cause of adult mortality worldwide. We have developed a rapidly adaptable platform to assess biomarkers using a microfluidic technology. Our model mimics autoantibodies against three proteins, C-reactive protein (CRP), brain natriuretic peptide (BNP), and low-density lipoprotein (LDL). Cell-free mitochondrial DNA (cfmDNA) and DNA controls are detected via fluorescence probes. The biomarkers are covalently bound on the surface of size- (11-15 μm) and dual-color encoded microbeads and immobilized as planar layer in a microfluidic chip flow cell. Binding events of target molecules were analyzed by fluorescence measurements with a fully automatized fluorescence microscope (end-point and real-time) developed in house. The model system was optimized for buffers and immobilization strategies of the microbeads to enable the simultaneous detection of protein and DNA biomarkers. All prime target molecules (anti-CRP, anti-BNP, anti-LDL, cfmDNA) and the controls were successfully detected both in independent reactions and simultaneously. In addition, the biomarkers could also be detected in spiked human serum in a similar way as in the optimized buffer system. The detection limit specified by the manufacturer is reduced by at least a factor of five for each biomarker as a result of the antibody detection and kinetic experiments indicate that nearly 50 % of the fluorescence intensity is achieved within 7 min. For rapid data inspection, we have developed the open source software digilogger, which can be applied for data evaluation and visualization. Graphical abstract.
快速同时检测 DNA 和蛋白质生物标志物对于检测疾病的爆发或监测疾病是必要的。例如，心血管疾病是全球成年人死亡的主要原因。我们已经开发出一种快速自适应平台，使用微流控技术评估生物标志物。我们的模型模拟了针对三种蛋白质（C 反应蛋白（CRP）、脑利钠肽（BNP）和低密度脂蛋白（LDL）的自身抗体。通过荧光探针检测无细胞线粒体 DNA（cfmDNA）和 DNA 对照。生物标志物通过共价键结合到大小为（11-15μm）的双色编码微珠表面，并固定在微流控芯片流动池的平面层中。通过自主开发的荧光显微镜（终点和实时）进行荧光测量分析靶分子的结合事件。该模型系统针对缓冲液和微珠的固定化策略进行了优化，以实现蛋白质和 DNA 生物标志物的同时检测。所有主要目标分子（抗 CRP、抗 BNP、抗 LDL、cfmDNA）和对照均在独立反应和同时反应中成功检测到。此外，还可以以类似的方式在优化的缓冲液系统中检测到掺入人血清中的生物标志物。由于抗体检测和动力学实验表明，每种生物标志物的检测限都至少降低了五倍，因此制造商规定的检测限降低了。为了快速数据检查，我们开发了开源软件 digilogger，可用于数据评估和可视化。图表摘要。
