Advanced Evanescent-Wave Optical Biosensors for the Detection of Nucleic Acids : An Analytic Perspective

Evanescent-wave optical biosensors have become an attractive alternative for the screening of nucleic acids in the clinical context. They possess highly sensitive transducers able to perform detection of a wide range of nucleic acid-based biomarkers without the need of any label or marker. These optical biosensor platforms are very versatile, allowing the incorporation of an almost limitless range of biorecognition probes precisely and robustly adhered to the sensor surface by covalent surface chemistry approaches. In addition, their application can be further enhanced by their combination with different processes, thanks to their integration with complex and automated microfluidic systems, facilitating the development of multiplexed and user-friendly platforms. The objective of this work is to provide a comprehensive synopsis of cutting-edge analytical strategies based on these label-free optical biosensors able to deal with the drawbacks related to DNA and RNA detection, from single point mutations assays and epigenetic alterations, to bacterial infections. Several plasmonic and silicon photonic-based biosensors are described together with their most recent applications in this area. We also identify and analyse the main challenges faced when attempting to harness this technology and how several innovative approaches introduced in the last years manage those issues, including the use of new biorecognition probes, surface functionalization approaches, signal amplification and enhancement strategies, as well as, sophisticated microfluidic solutions

Simple, low-cost and timely optical biosensors for the detection of epigenetics biomarkers : the future of cancer diagnosis

The cancer burden continues to grow with enormous physical, emotional, and financial pressure on individuals, families, communities, and health systems. Early detection and effective treatment are crucial. The analysis of epigenetic biomarkers is presented as an exceptional solution for early cancer diagnosis and personalised treatment design. These brand new biomarkers have initiated a diagnostic revolution because of their predictive capability and reversibility, opening the window for timely diagnostics and personalised medicine. In recent years, the potential of optical biosensors for epigenetic biomarker evaluation has been revealed. Nanotechnology is promoting the appearance of new advanced biosensors able to be integrated in complete lab-on-chip platforms. Lab-on-chip biosensors are offering simplified, cost-effective, and fast results to solve the current diagnostic problems. In this review, we present the advantages offered by the analysis of epigenetic routes in cancer diagnosis and the current advances in optical biosensors for cancer epigenetic analysis, showing how the new biosensor solutions manage to surpass the challenges encountered during the analysis of each epigenetic mechanism

Simultaneous time-space resolved reflectivity and interferometric measurements of dielectrics excited with femtosecond laser pulses

13 pags., 7 figs., 1 tab.Simultaneous time-and-space resolved reflectivity and interferometric measurements over a temporal span of 300 ps have been performed in fused silica and sapphire samples excited with 800 nm, 120 fs laser pulses at energies slightly and well above the ablation threshold. The experimental results have been simulated in the frame of a multiple-rate equation model including light propagation. The comparison of the temporal evolution of the reflectivity and the interferometric measurements at 400 nm clearly shows that the two techniques interrogate different material volumes during the course of the process. While the former is sensitive to the evolution of the plasma density in a very thin ablating layer at the surface, the second yields an averaged plasma density over a larger volume. It is shown that self-trapped excitons do not appreciably contribute to carrier relaxation in fused silica at fluences above the ablation threshold, most likely due to Coulomb screening effects at large excited carrier densities. For both materials, at fluences well above the ablation threshold, the maximum measured plasma reflectivity shows a saturation behavior consistent with a scattering rate proportional to the plasma density in this fluence regime. Moreover, for both materials and for pulse energies above the ablation threshold and delays in the few tens of picoseconds range, a simultaneous >low reflectivity> and >low transmission> behavior is observed. Although this behavior has been identified in the past as a signature of femtosecond laser-induced ablation, its origin is alternatively discussed in terms of the optical properties of a material undergoing strong isochoric heating, before having time to substantially expand or exchange energy with the surrounding media.This work has been partly funded by Laserlab-Europe (Grant Agreement No. 284464, EU’s Seventh Framework Programme, Project No. SLIC002014), by the Spanish Ministry of Economy and Competiveness (Project No. TEC2014-52642- C2-1-R) as well as by the Danish Council for Independent Research | Natural Sciences. M.G.-L. acknowledges the FPU (Formación de Profesorado Universitario) Grant No. AP2012- 0217 awarded by the Spanish Ministry of Education.Peer Reviewe

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

Analysis of alternative splicing events for cancer diagnosis using a multiplexing nanophotonic biosensor

Personalized medicine is a promising tool not only for prevention, screening and development of more efficient treatment strategies, but also for diminishing the side effects caused by current therapies. Deciphering gene regulation pathways provides a reliable prognostic analysis to elucidate the origin of grave diseases and facilitate the selection of the most adequate treatment for each individual. Alternative splicing of mRNA precursors is one of these gene regulation pathways and 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 constituting a relevant and innovative class of biomarker. Herein we report a highly selective and sensitive nanophotonic biosensor based on the direct monitoring of the aberrant alternative splicing of Fas gene. Unlike conventional methods, the nanobiosensor performs a real-time detection of the specific isoforms in the fM-pM range without any cDNA synthesis or PCR amplification requirements. The nanobiosensor has been proven isoform-specific with no crosshybridization, greatly minimizing detection biases. The demonstrated high sensitivity and specificity make our nanobiosensor ideal for examining significant tumor-associated expression shifts of alternatively spliced isoforms for the early and accurate theranostics of cancer

Label-free plasmonic biosensors for point-of-care diagnostics : a review

Introduction: Optical biosensors, particularly those based on nanoplasmonics technology, have emerged in recent decades as a potential solution for disease diagnostics and therapy follow-up at the point-of-care (POC). These biosensor platforms could overcome some of the challenges faced in conventional diagnosis techniques offering label-free assays with immediate results and employing small and user-friendly devices. Areas covered: In this review, we will provide a critical overview of the recent advances in the development of nanoplasmonic biosensors for the POC diagnostics. We focus on those systems with demonstrated capabilities for integration in portable platforms, highlighting some of the most relevant diagnostics applications targeting proteins, nucleic acids, and cells as disease biomarkers. Expert commentary: Despite the attractive features of label-free nanoplasmonic sensors in terms of miniaturization and analytical robustness, the route toward an effective clinical implementation involves the integration of fully automated microfluidic systems for sample processing and analysis, and the optimization of surface biofunctionalization procedures. Additionally, the development of multiplexed sensors for high-throughput analysis and including specific neoantigens and novel biomarkers in detection panels will provide the means for delivering a powerful analytical technology for an accurate and improved medical diagnosis

Prospects of optical biosensors for emerging label-free RNA analysis

RNA is critical in countless cellular processes, and researchers are constantly discovering new types and attributing them different roles. Consequently, a growing interest in efficient RNA analysis has arisen. However, RNA detection is complicated and generally requires the use of labels. Major efforts are being devoted to conceive new approaches for RNA analysis with no need of markers. Optical biosensing is a highly sensitive approach that circumvents many of conventional methods' limitations. Lately, label-free applications with optical biosensors have been developed for short as well as for long RNAs. The low limits of detection at the pM level enabled by optical biosensors, together with a fast analysis, their reusability and the label-free scheme of operation, clearly highlight them among the most promising next-generation RNA screening platforms. This review covers the most relevant optical biosensor platforms and their potential for enabling sensitive and label-free RNA analysis

Sensitive and label-free detection of miRNA-145 by triplex formation

The development of new strategies for detecting microRNAs (miRNAs) has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work, we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and circular dichroism (CD) measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a surface plasmon resonance (SPR) biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes