Detection of miRNA cancer biomarkers using light activated Molecular Beacons

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. Photonic based detection strategies of cancer miRNA biomarkers after Light Assisted Molecular Immobilization (LAMI) of peptide-MB biosensor constructs.