Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing.

Alternative splicing of mRNA precursors enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression. Current methodologies for monitoring alternative splicing demand elaborate procedures and often present difficulties in discerning between closely related isoforms, e.g. due to cross-hybridization during their detection. Herein, we report a general methodology using a Surface Plasmon Resonance (SPR) biosensor for label-free monitoring of alternative splicing events in real-time, without any cDNA synthesis or PCR amplification requirements. We applied this methodology to RNA isolated from HeLa cells for the quantification of alternatively spliced isoforms of the Fas gene, involved in cancer progression through regulation of programmed cell death. We demonstrate that our methodology is isoform-specific, with virtually no cross-hybridization, achieving limits of detection (LODs) in the picoMolar (pM) range. Similar results were obtained for the detection of the BCL-X gene mRNA isoforms. The results were independently validated by RT-qPCR, with excellent concordance in the determination of isoform ratios. The simplicity and robustness of this biosensor technology can greatly facilitate the exploration of alternative splicing biomarkers in disease diagnosis and therapy.

The effects of lipids and surfactants on TLR5-proteoliposome functionality for flagellin detection using surface plasmon resonance biosensing.

The use of proteoliposomes as affinity elements in conjunction with a surface plasmon resonance sensor is a high-sensitivity alternative for the detection of multiple analytes. However, one of the most important aspects of these conformations is maintaining the functionality of the immobilized protein, which is determined by the choice of lipids and surfactants employed in the reconstitutions. Previously, we demonstrated the functionality of TLR5-proteoliposomes as screening affinity elements of bacterial flagellin. In this new study we change the conditions of immobilization of TLR5 and evaluate how the fluidity of the membrane and the final size of the liposomes affect the functionality of the construct and thus increase their utility as an affinity element for design of new biosensors. In particular, we used reconstructions into preformed liposomes composed of the lipids POPC, POPC-DMPC and POPC-POPE mediated by the use of surfactants OG, Triton X100, and DDM, respectively. The affinity results were evaluated by SPR technology proteoliposomes and were correlated with the anisotropic change in the membrane status; the final sizes of the proteoliposomes were estimated. Our results clearly show the dependence of fluidity and final size of the proteoliposomes with surface plasmon resonance affinity measurements.

Detection of flagellin by interaction with human recombinant TLR5 immobilized in liposomes.

Digestive diseases caused by flagellated bacteria are a huge public health problem worldwide and rapid detection methods are needed for contaminated environments. In this study, we propose a method to detect patterns associated with pathogens based on the properties of the innate immune system. Specifically, we use Toll-like receptor 5 (TLR5), a transmembrane protein that specifically recognizes flagellin (the structural protein of bacterial flagella). TLR5, which was obtained by recombinant production in insect cells, was immobilized into liposomes to form TLR5-proteoliposomes. Through surface plasmon resonance (SPR) and competition flow cytometry assays, the sensitivity of proteoliposomes to recognize Escherichia coli and Salmonella typhimurium flagellin was evaluated. In addition, we compared the results obtained by immobilizing anti-flagellin antibodies into liposomes. The results of the flagellin-affinity tests, expressed as an SPR kinetic rate constant ratio in the equilibrium equation K(D) = k(d)/k(a), showed values of 13.8 × 10(-9) and 7.73 × 10(-9) M for the TLR5-proteoliposomes and anti-flagellin antibodies, respectively, against S. typhimurium. The anti-flagellin affinity results for E. coli showed K(D) of 84.1 × 10(-8) M for SPR assays and K (D) of 3.5 × 10(-8) M for competitive flow cytometry, which was used as a detection system without the immobilization of proteoliposomes. This research demonstrates the practical possibility of using proteoliposomes as recognition elements in the generation of systems for the rapid detection of flagellated bacteria, which could help avoid consumption of contaminated food by humans and thereby prevent intestinal infections.

Label-free detection of DNA mutations by SPR: application to the early detection of inherited breast cancer.

A screening analysis of DNA hybridization and the presence of DNA mutations using an surface plasmon resonance (SPR) biosensor is shown. The influence of lateral and vertical spacers, as well as several hybridization conditions, was studied to optimize the differentiation between fully complementary and mismatched DNA strands. Our results demonstrated that SPR biosensors were able to detect mismatch sequences related to inherited breast cancer, with high specificity and sensitivity. Using PCR synthetic sequences as targets, mutant sequences were clearly discriminated from fully complementary ones, and detection limits below 50 nM were achieved.

Nanomechanics of the formation of DNA self-assembled monolayers and hybridization on microcantilevers.

Biomolecular interactions over the surface of a microcantilever can produce its bending motion via changes of the surface stress, which is referred to nanomechanical response. Here, we have studied the interaction forces responsible for the bending motion during the formation of a self-assembled monolayer of thiolated 27-mer single-stranded DNA on the gold-coated side of a microcantilever and during the subsequent hybridization with the complementary nucleic acid. The immobilization of the single-stranded DNA probe gives a mean surface stress of 25 mN/m and a mean bending of 23 nm for microcantilevers with a length and thickness of about 200 microm and 0.8 microm, respectively. The hybridization with the complementary sequence could not be inferred from the nanomechanical response. The nanomechanical response was compared with data from well-established techniques such as surface plasmon resonance and radiolabeling, to determine the surface coverage and study the intermolecular forces between neighboring DNA molecules anchored to the microcantilever surface. From both techniques, an immobilization surface density of 3 x 10(12) molecules/cm(2) and a hybridization efficiency of 40% were determined. More importantly, label-free hybridization was clearly detected in the same conditions with a conventional sensor based on surface plasmon resonance. The results imply that the nanomechanical signal during the immobilization process arises mainly from the covalent attachment to the gold surface, and the interchain interactions between neighboring DNA molecules are weak, producing an undetectable surface stress. We conclude that detection of nucleic acid hybridization with nanomechanical sensors requires reference cantilevers to remove nonspecific signals, more sensitive microcantilever geometries, and immobilization chemistries specially addressed to enhance the surface stress variations.