Department of Biochemistry & Molecular Medicine, University of California Davis, Sacramento, CA 95817, USA.
Department of Biomedical Engineering, University of California Davis, Davis, CA 95616, USA.
Molecules. 2019 May 10;24(9):1813. doi: 10.3390/molecules24091813.
The United States is currently experiencing an opioid crisis, with more than 47,000 deaths in 2017 due to opioid overdoses. Current approaches for opioid identification and quantification in body fluids include immunoassays and chromatographic methods (e.g., LC-MS, GC-MS), which require expensive instrumentation and extensive sample preparation. Our aim was to develop a portable point-of-care device that can be used for the instant detection of opioids in body fluids. Here, we reported the development of a morphine-sensitive fluorescence-based sensor chip to sensitively detect morphine in the blood using a homogeneous immunoassay without any washing steps. Morphine-sensitive illuminating peptides were identified using a high throughput one-bead one-compound (OBOC) combinatorial peptide library approach. The OBOC libraries contain a large number of random peptides with a molecular rotor dye, malachite green (MG), that are coupled to the amino group on the side chain of lysine at different positions of the peptides. The OBOC libraries were then screened for fluorescent activation under a confocal microscope, using an anti-morphine monoclonal antibody as the screening probe, in the presence and absence of free morphine. Using this novel three-step fluorescent screening assay, we were able to identify the peptide-beads that fluoresce in the presence of an anti-morphine antibody, but lost fluorescence when the free morphine was present. After the positive beads were decoded using automatic Edman microsequencing, the morphine-sensitive illuminating peptides were then synthesized in soluble form, functionalized with an azido group, and immobilized onto microfabricated PEG-array spots on a glass slide. The sensor chip was then evaluated for the detection of morphine in plasma. We demonstrated that this proof-of-concept platform can be used to develop fluorescence-based sensors against morphine. More importantly, this technology can also be applied to the discovery of other novel illuminating peptidic sensors for the detection of illicit drugs and cancer biomarkers in body fluids.
美国目前正经历一场阿片类药物危机，2017 年有超过 4.7 万人死于阿片类药物过量。目前用于鉴定和定量体液中阿片类药物的方法包括免疫测定和色谱方法（例如 LC-MS、GC-MS），这些方法需要昂贵的仪器和广泛的样品制备。我们的目标是开发一种便携式即时检测设备，可用于即时检测体液中的阿片类药物。在这里，我们报告了一种基于吗啡敏感荧光的传感器芯片的开发，该芯片通过均相免疫测定法在无需任何洗涤步骤的情况下灵敏地检测血液中的吗啡。使用高通量的单珠单化合物（OBOC）组合肽文库方法鉴定出对吗啡敏感的发光肽。OBOC 文库包含大量随机肽，其分子转子染料孔雀石绿（MG）与肽侧链赖氨酸上的氨基在不同位置偶联。然后，在存在和不存在游离吗啡的情况下，使用抗吗啡单克隆抗体作为筛选探针，在共聚焦显微镜下筛选 OBOC 文库，以检测荧光激活。使用这种新颖的三步荧光筛选测定法，我们能够识别出在存在抗吗啡抗体时发出荧光的肽珠，但当存在游离吗啡时则失去荧光。在用自动 Edman 微测序解码阳性珠后，然后将吗啡敏感的发光肽以可溶性形式合成，官能化叠氮基团，并固定在玻璃载玻片上的微制造 PEG 阵列点上。然后评估传感器芯片对血浆中吗啡的检测。我们证明了这个概念验证平台可用于开发针对吗啡的荧光传感器。更重要的是，这项技术还可应用于发现其他新型发光肽类传感器，用于检测体液中的非法药物和癌症生物标志物。
