Site-Specific mRNA Cleavage for Selective and Quantitative Profiling of Alternative Splicing with Label-Free Optical Biosensors.

Alternative splicing of mRNA precursors is a key process in gene regulation, contributing to the diversity of proteomes by the alternative selection of exonic sequences. Alterations in this mechanism are associated with most cancers, enhancing their proliferation and survival, and can be employed as cancer biomarkers. Label-free optical biosensors are ideal tools for the highly sensitive and label-free analysis of nucleic acids. However, their application for alternative splicing analysis has been hampered due to the formation of complex and intricate long-range base-pairing interactions which make the direct detection in mRNA isoforms difficult. To solve this bottleneck, we introduce a methodology for the generation of length-controlled RNA fragments from purified total RNA, which can be easily detected by the biosensor. The methodology seizes RNase H enzyme activity to degrade the upstream and downstream RNA segments flanking the target sequence upon hybridization to specific DNA oligos. It allows the fast and direct monitoring of Fas gene alternative splicing in real time, employing a surface plasmon resonance biosensor. We demonstrate the selective and specific detection of mRNA fragments in the pM-nM concentration range, reducing quantification errors and showing 81% accuracy when compared to RT-qPCR. The site-specific cleavage outperformed random RNA hydrolysis by increasing the detection accuracy by 20%, making this methodology particularly appropriate for label-free quantification of alternative splicing events in complex samples.

Development of a surface plasmon resonance and nanomechanical biosensing hybrid platform for multiparametric reading.

We have developed a hybrid platform that combines two well-known biosensing technologies based on quite different transducer principles: surface plasmon resonance and nanomechanical sensing. The new system allows the simultaneous and real-time detection of two independent parameters, refractive index change (Δn), and surface stress change (Δσ) when a biomolecular interaction takes place. Both parameters have a direct relation with the mass coverage of the sensor surface. The core of the platform is a common fluid cell, where the solution arrives to both sensor areas at the same time and under the same conditions (temperature, velocity, diffusion, etc.).The main objective of this integration is to achieve a better understanding of the physical behaviour of the transducers during sensing, increasing the information obtained in real time in one single experiment. The potential of the hybrid platform is demonstrated by the detection of DNA hybridization.

Indirect competitive immunoassay for the detection of fungicide Thiabendazole in whole orange samples by Surface Plasmon Resonance.

A highly sensitive and specific SPR-based competitive immunoassay for the detection of Thiabendazole (TBZ) has been developed. An indirect format where a TBZ-protein conjugate is immobilized onto gold surfaces has been selected. Under the optimal conditions, a LOD of 0.67 nM (0.13 µg L(-1)) and an IC(50) of 3.2 nM (0.64 µg L(-1)) have been achieved which are comparable to the values obtained by conventional ELISA. Analysis of real samples has been attempted by first evaluating the influence of complex matrix samples coming from whole oranges and secondly measuring samples containing TBZ previously evaluated by chromatographic methods. A methanolic extraction procedure followed by a simple dilution in assay buffer has proven to be sufficient to measure orange samples using the developed immunoassay with an excellent recovery percentage. The sensitivity and the feasibility of measuring whole orange samples demonstrate the effectiveness and robustness of the SPR biosensor, which can be useful for the determination of TBZ in food at concentrations below the Maximum Residue Levels (MRLs) established by the European legislation.