Abstract:A label-free sensor, based on the combination of silicon photonic bandgap (PBG) structures with immobilized molecular beacon (MB) probes, is experimentally developed. Complementary target oligonucleotides are specifically recognized through hybridization with the MB probes on the surface of the sensing structure. This combination of PBG sensing structures and MB probes demonstrates an extremely high sensitivity without the need for complex PCR-based amplification or labelling methods. correlates with morphological sub-class of acute myeloid leukaemia and the expression of its target genes in global genome-wide analysis," Leukemia 21(5), 912-916 (2007). 4. R. L. Maute, C. Schneider, P. Sumazin, A. Holmes, A. Califano, K. Basso, and R. Dalla-Favera, "tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma," Proc. Natl. Acad. Sci. U.S.A. 110(4), 1404-1409 (2013). 5. L. P. Lim, N. C. Lau, P. Garrett-Engele, A. Grimson, J. M. Schelter, J. Castle, D. P. Bartel, P. S. Linsley, and J.M. Johnson, "Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs," Nature 433(7027), 769-773 (2005

An experimental study of the influence of the conformational change suffered by molecular beacon (MB) probes-upon the biorecognition of nucleic acid target oligonucleotides over evanescent wave photonic sensors-is reported. To this end, high sensitivity photonic sensors based on silicon photonic bandgap (PBG) structures were used, where the MB probes were immobilized via their 5' termination. Those MBs incorporate a biotin moiety close to their 3' termination in order to selectively bind a streptavidin molecule to them. The different photonic sensing responses obtained toward the target oligonucleotide detection, when the streptavidin molecule was bound to the MB probes or not, demonstrate the conformational change suffered by the MB upon hybridization, which promotes the displacement of the streptavidin molecule away from the surface of the photonic sensing structure.

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 labelfree 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.Fig. 4 Photonic bandgap (PBG) sensing structures. (Left) Schematic representation of the PBG sensing structure used to carry out the photonic miRNA sensing experiments. (Right) Scanning Electron Microscope (SEM) image of one of the PBG sensing structures fabricated in this work. This journal isView Article Online a Y offset (y 0 ), amplitude (A i ), rate constants (t i ), intensity fraction (f i ) and average rate constant (hti) for hybridization of poly-A and miRNA-21 specic MB constructs with the respective complementary oligonucleotides (ratios 1 : 1 and 1 : 10).This journal is

The optimal development of biosensors is a costly and time-consuming task, since an enormous amount of experiments is required. Therefore, the possibility of reusing the biosensors is highly desirable. In this work, a protocol based on the use of formamide for the regeneration of nanophotonic biosensors used for oligonucleotides detection is presented. This protocol was carried out online using the microfluidic system used to drive the target samples to the nanophotonic biosensor, thus allowing the possibility of running several experiments in a row using the same biosensor.