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Detection of miRNA cancer biomarkers using light activated Molecular Beacons

机译：使用光活性分子信标检测miRNA癌生物标志物

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摘要

Early detection of cancer biomarkers can reduce cancer mortality rate. miRNAs are small non-coding RNAs whose expression changes upon the onset of various types of cancer. Biosensors that specifically detect such biomarkers can be engineered and integrated into point-of-care devices (POC) using label-free detection, high sensibility and compactness. In this paper, a new engineered Molecular Beacon (MB) construct used to detect miRNAs is presented. Such a construct is immobilized onto biosensor surfaces in a covalent and spatially oriented way using the photonic technology Light Assisted Molecular Immobilization (LAMI). The construct consists of a Cy3 labelled MB covalently attached to a light-switchable peptide. One MB construct contains a poly-A sequence in its loop region while the other contains a sequence complementary to the cancer biomarker miRNA-21. The constructs have been characterized by UV-Vis spectroscopy, mass spectrometry and HPLC. LAMI led to the successful immobilization of the engineered constructs onto thiol functionalized optically flat quartz slides and Silicon on Insulator (SOI) sensor surfaces. The immobilized Cy3 labelled MB construct has been imaged using confocal fluorescence microscopy (CFM). The bioavailability of the immobilized engineered MB biosensors was confirmed through specific hybridization with the Cy5 labelled complementary sequence and imaged by CFM and FRET. Hybridization kinetics have been monitored using steady state fluorescence spectroscopy. The label-free detection of miRNA-21 was also achieved by using integrated photonic sensing structures. The engineered light sensitive constructs can be immobilized onto thiol reactive surfaces and are currently being integrated in a POC device for the detection of cancer biomarkers.
机译：早期发现癌症生物标志物可以降低癌症死亡率。 miRNA是小的非编码RNA，其表达在各种类型的癌症的开始时变化。专门检测这种生物标志物的生物传感器可以使用无标签检测，高敏感性和紧凑性来设计和整合到护理点装置（POC）中。本文介绍了用于检测miRNA的新的工程分子信标（MB）构建体。将这种构建体固定在生物传感器表面上，以共价和空间取向的方式，使用光子技术光辅助分子固定（Lami）。构建体由标记的Mb共价连接到可轻可切换肽的Cy3组成。一个MB构建体含有环形区域中的聚序列，而另一个含有与癌症生物标志物miRNA-21互补的序列。通过UV-Vis光谱，质谱和HPLC表征了构建体。 Lami导致将工程化构造成功固定到硫醇官能化的光学平石英载玻片上和绝缘体上的硅（SOI）传感器表面上。通过共聚焦荧光显微镜（CFM）对固定的Cy3标记的MB构建体进行了成像。通过特异性杂交证实固定化的工程化MB生物传感器的生物利用度与标记的互补序列标记的互补序列和CFM和FRET成像。使用稳态荧光光谱监测杂交动力学。通过使用集成的光子传感结构，还实现了MiRNA-21的无标记检测。工程化的光敏感构建体可以固定在硫醇反应表面上，并且目前正在集成在POC装置中以检测癌症生物标志物。

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  • 来源
    《RSC Advances》 |2019年第22期|共18页
  • 作者

    Goncalves Odete Sofia Lopes; Wheeler Guy; Dalmay Tamas; Dai Houquan; Castro Miguel; Castro Patrick; Garcia-Ruperez Jaime; Ruiz-Tortola Angela; Griol Amadeu; Hurtado Juan; Bellieres Laurent; Jose Banuls Maria; Gonzalez Daniel; Antonio Lopez-Guerrero Jose; Neves-Petersen Maria Teresa;

  • 作者单位

    Aalborg Univ Fac Med Dept Hlth Sci &

    Technol Med Photon Lab Fredrik Bajers Vej 7 DK-9220 Aalborg Denmark;

    Univ East Anglia Sch Biol Sci Norwich Res Pk Norwich NR4 7TJ Norfolk England;

    Univ East Anglia Sch Biol Sci Norwich Res Pk Norwich NR4 7TJ Norfolk England;

    Biosynthesis 612 East Main St Lewisville TX 75057 USA;

    Biosynthesis 612 East Main St Lewisville TX 75057 USA;

    Biosynthesis 612 East Main St Lewisville TX 75057 USA;

    Univ Politecn Valencia Nanophoton Technol Ctr Camino Vera S-N E-46022 Valencia Spain;

    Univ Politecn Valencia Nanophoton Technol Ctr Camino Vera S-N E-46022 Valencia Spain;

    Univ Politecn Valencia Nanophoton Technol Ctr Camino Vera S-N E-46022 Valencia Spain;

    Univ Politecn Valencia Nanophoton Technol Ctr Camino Vera S-N E-46022 Valencia Spain;

    Univ Politecn Valencia Nanophoton Technol Ctr Camino Vera S-N E-46022 Valencia Spain;

    Univ Politecn Valencia Dept Quim IDM Inst Interuniv Invest Reconocimiento Mol &

    Desarr Camino Vera S-N E-46022 Valencia Spain;

    Univ Politecn Valencia Dept Quim IDM Inst Interuniv Invest Reconocimiento Mol &

    Desarr Camino Vera S-N E-46022 Valencia Spain;

    Fdn Inst Valenciano Oncol Biol Mol Valencia Spain;

    Aalborg Univ Hosp Dept Clin Med Hobrovej 18-22 DK-9000 Aalborg Denmark;

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