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

Abstract

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.

Keywords: biosensors; clinical diagnosis; epigenetics; microfluidics; nucleic acid analysis; plasmonics; silicon photonics; surface chemistry.

Figure 1

Different label-free optical biosensor based on evanescent wave. (A) Scheme of a biosensor. Reprinted from Carrascosa et al. (2016), Copyright (2018), with permission from Elsevier. (B) Evanescent field principle. (C) SPR biosensor based on Krestchman configuration and different detection modes: (i) fixed-angle, (ii) fixed wavelength, and (iii) fixed angle and wavelength (SPRi). (D) LSPR light coupling methods: (i) prism, (ii) extinction, and (iii) dark field. (E) Micro-ring resonator, (F) MZI biosensor, and (G) BiMW biosensor designs and working principles.

Figure 2

Nucleic-acid biosensors surface functionalization. (A) Scheme of a standard DNA probe. (B) Different surface coverages: (i) low, (ii) high, and (iii) mixed monolayer. (C) Gold surface immobilization strategies based on direct chemisorption (left) and on the generation of a functional layer (right). (D) Silicon surface immobilization strategies through silanes without (left) or with (right) crosslinkers.

Figure 3

Amplification strategies. (A) Enzymatic reaction. Nuclease digests the probe and the target can be recycled to hybridize with another probe. (B) Self-catalytic reaction. Hybridization chain reaction starts when target hybridizes with the hairpin probe. It triggers the coupling of two partially complementary DNA sequences called DNA helpers. (C) Protein binding nucleic acids. Anti-DNA-RNA antibody recognizes DNA-RNA hybrids. (D) Nanomaterials. Functionalized AuNPs generate DNA sandwich structures with the hybrids.

Figure 4

Detection of SNPs and methylation profile with optical biosensors. (A) Detection of two SNPs in KRAS gene using silicon microring resonators sensors. (i) ISAD-KRAS assay representation and calibration curves reflecting the resonant wavelength shift obtained for G12D and G13D mutants of KRAS gene (gray: wild-type, black: G12D mutant, red: G13D mutant). (ii) Validation of the ISAD-KRAS assay in 70 clinical samples of colorectal cancer patients (50 mutants and 20 wild-type) for G12D and G13D mutations, and comparison with conventional techniques as PCR and sequencing. Purple oval represents the mutant area and yellow oval the wild-type one. The mutant allele (black) and wild allele (blue) are also shown. Readapted with permission from Jin et al. (2017). Copyright © 2017 Jin et al. Creative Commons Attribution License 3.0 (CC BY 3.0). (B) Detection of the methylation profile of PAX-5 gene using a SPR biosensor. (i) Scheme and real-time recognition of the two-step assay developed for the detection of (1) ds-DNA fragments and (2) 5'methyl cytosines by the PPRH probe and specific anti-5-mC antibody, respectively. (ii) Identification of different methylation profiles (0x, 1x, and 4x 5' methyl cytosines). Reprinted from Huertas et al. (2018), Copyright (2018), with permission from Elsevier.

Figure 5

Detection of long RNAs with optical biosensors. (A) Quantification of alternatively spliced mRNA isoforms from Fas gene using a SPR biosensor. (i) Scheme of the different RNA isoforms generated by alternative splicing of Fas gene. (ii) Calibration curves for Fas57 and Fas567 isoforms and scheme of the DNA-probes used for the hybridization. (iii) SPR sensograms of the detection of total extracted HeLa cell RNA for Fas56 (left) and Fas57 (right) probes. Reprinted from Huertas et al. (2016a), Copyright (2016), with permission from Elsevier. (B) Detection of 16S rRNA from Pseudomonas aeruginosa, Salmonella typhimurium, and Legionella pneumophila using a SPRi sensor. (i) Capture probes (CP) are immobilized for the hybridization of RNA and amplification is performed by using nanoparticles functionalized with the detection probes (GNP-DP). Probe strategy design are illustrated using 16S rRNA sequence of Pseudomonas aeruginosa as an example. (ii) SPR sensograms and calibration curves for quantification of L. pneumophila RNA strain using amplification. Adapted with permission from Melaine et al. (2017). Copyright © 2017 American Chemical. (C) Detection of KIAA0495 and MALAT1 lncRNAs with microring resonator arrays. (i) Scheme of the overall assay for lncRNA detection. (ii) Identification of each lncRNA and evaluation of the specificity using β-actin as internal control. (iii) Assessment of lncRNAs expression in GBM6 cells compared to conventional, single-plex RT-qPCR technique. Healthy brain (1) and lung (2) tissues were used as reference. Republished with permission of The Royal Society of Chemistry from Cardenosa-Rubio et al. (2018); permission conveyed through Copyright Clearance Center Inc.

Figure 6

Detection of miRNAs by optical biosensors. (A) Detection of miR-10b for LSPR-based biosensor. (i) Schematic representation of miRNA detection using duplex-specific nuclease for recycling and hybridization chain reaction with tannic acid tags for amplification. (ii) LSPR shifts in urine and plasma mice with orthotopic Hs746tT xenografts samples compared to normal model mice. Reprinted with permission from Ki et al. (2019). Copyright © 2019, American Chemical Society. (B) Detection of miR-181a using a BiMW interferometer. (i) Working principle of the BiMW interferometer and biofunctionalization strategy. (ii) Calibration curve of miR-181a in semilog scale. (iii) miR-181a quantification in urine samples from healthy donors and bladder cancer patients. Adapted with permission from Huertas et al. (2016b). Copyright © 2016, American Chemical Society.