REVIEW published: 25 October 2019 doi: 10.3389/fchem.2019.00724 Advanced Evanescent-Wave Optical Biosensors for the Detection of Nucleic Acids: An Analytic Perspective Cesar S. Huertas 1*, Olalla Calvo-Lozano 2 , Arnan Mitchell 1 and Laura M. Lechuga 2 1 Integrated Photonics and Applications Centre, School of Engineering, Royal Melbourne Institute of Technology University, Melbourne, VIC, Australia, 2 Nanobiosensors and Bioanalytical Applications Group, Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and the Barcelona Institute of Science and Technology, CIBER-BBN, Barcelona, Spain 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 Edited by: and automated microfluidic systems, facilitating the development of multiplexed and Ilaria Palchetti, user-friendly platforms. The objective of this work is to provide a comprehensive synopsis University of Florence, Italy of cutting-edge analytical strategies based on these label-free optical biosensors able to Reviewed by: Xiaohong Zhou, deal with the drawbacks related to DNA and RNA detection, from single point mutations Tsinghua University, China assays and epigenetic alterations, to bacterial infections. Several plasmonic and silicon Hui Xu, photonic-based biosensors are described together with their most recent applications Ludong University, China in this area. We also identify and analyse the main challenges faced when attempting *Correspondence: Cesar S. Huertas to harness this technology and how several innovative approaches introduced in the cesar.sanchez.huertas@rmit.edu.au last years manage those issues, including the use of new biorecognition probes, surface functionalization approaches, signal amplification and enhancement strategies, as well Specialty section: This article was submitted to as, sophisticated microfluidic solutions. Analytical Chemistry, Keywords: biosensors, plasmonics, silicon photonics, microfluidics, nucleic acid analysis, epigenetics, surface a section of the journal chemistry, clinical diagnosis Frontiers in Chemistry Received: 13 August 2019 Accepted: 10 October 2019 Published: 25 October 2019 INTRODUCTION Citation: Nucleic acids (NA) have a key function in many important cellular mechanisms, such as cell Huertas CS, Calvo-Lozano O, differentiation, cell-division cycle, signal transduction, or metabolism (Fatica and Bozzoni, 2014; Mitchell A and Lechuga LM (2019) Mens and Ghanbari, 2018; Mori, 2018). Our DNA is the best-known NA molecule and it contains Advanced Evanescent-Wave Optical Biosensors for the Detection of our genetic information. It is translated into proteins by the expression of messenger RNA Nucleic Acids: An Analytic (mRNAs). However, this translation may be more complex than faithful transcription of the DNA Perspective. Front. Chem. 7:724. code to form proteins. Indeed, many species have cellular mechanisms that can edit the mRNAs by doi: 10.3389/fchem.2019.00724 alternative splicing processes, resulting in a significant increase in protein diversity (Liu et al., 2012). Frontiers in Chemistry | www.frontiersin.org 1 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors The gene regulation process is made even more complex Also, RG108, which is an inhibitor of DNA methylation enzymes, by the existence of a series of epigenetic mechanisms which can suppress radio-resistance in esophageal cancer patients (Ou produce a phenotypic alteration in the expression of our genome et al., 2018). Likewise, several small molecules that regulate RNA while keeping intact the DNA code. Among these epigenetic splicing have been acknowledged as good drug candidates for processes can be found: the methylation of the DNA, nucleosome treatment (Salton and Misteli, 2016), and personalized RNA- repositioning, post-translational modification of histones, and based therapy has been proposed to decrease tumor growth in post-transcriptional gene regulation by non-coding RNAs (nc- pancreatic cancer (Gilles et al., 2018). RNAs), such as micro-RNAs (miRNAs) (Yang et al., 2018). The most widely applied NA detection methods are those This set of genetic and epigenetic mechanisms results in a based on Polymerase Chain Reaction (PCR) or sequencing highly-structured gene regulatory network that serves the cells techniques (Cheng et al., 2018; Elazezy and Joosse, 2018; Zhao, to meet their specific function and create opportunities to 2019). PCR-based approaches allow the examination of small overcome environmental changes within the lifetime of an quantities of material with high sensitivity in a time range from individual organism. few minutes to hours, which has greatly promoted the clinical In recent years, the analysis of NA for diagnostic purposes analysis of NA molecules. PCR-based approaches have enabled have received special attention because of their prospective the identification of specific NA sequence variations, such as: application as biomarkers for the early and accurate detection point mutations or single nucleotide polymorphisms (SNPs) of infectious diseases (Fournier et al., 2013) and the molecular (Zonta et al., 2016); DNA methylation (Fackler and Sukumar, diagnosis of a great number of clinical disorders, and in particular 2018); alternative splicing variants (Harvey and Cheng, 2016); cancer (Gingeras et al., 2005; Asadollahi et al., 2010; Lo et al., miRNAs (Wang et al., 2019); as well as bacterial resistance 2010; Jansson and Lund, 2012; Benesova et al., 2013; Oltean and (Schmidt et al., 2019). On the other hand, next generation Bates, 2013). Fortunately, NA biomarkers have been found to sequencing (NGS) methods can perform genome-wide or exon- circulate in diverse biological media, including serum, plasma, wide analysis by massive parallel sequencing, allowing the saliva, and urine (Pös et al., 2018), so they can be targeted in screening of copy number aberrations (Belic et al., 2016) or the development of new diagnostic approaches. For example, SNPs (Gale et al., 2018). They have also been employed for the circulating NAs are employed as target biomarkers in liquid identification of methylation sites (Wen et al., 2015). Sequencing biopsies in substitution of the hazardous tissue biopsies. This approaches enable to detect sequence variations that may take could overcome current clinical limitations in encompassing the place during the cancer treatment cycle without any previous cancer cell genome and epigenome heterogeneity over time since evidence of the primary tumour’s genome. In general, the a liquid biopsy allows the recurrent monitoring of blood samples application of these methodologies for a rapid and efficient in a less invasive manner. The information gathered from these population screening still face some limitations, such as the time biomarkers can provide better insight into the evolutionary to complete the analysis, which usually ranges from days to hours dynamics of cancer. In fact, circulating tumor DNA (ctDNA) for DNA sequencing and PCR-based, respectively, and the large seems to be a better indicator of the patient tumor status when volumes of sample required (Bellassai and Spoto, 2016). compared with clinical serological markers (Sanmamed et al., Considering these facts, efforts should be directed into 2015; Chang et al., 2016; Girotti et al., 2016). Other circulating the development of new technologies that enable the efficient NAs such as mRNA and miRNA offer other level of information detection of genetic and epigenetic biomarkers involving simpler (Climente-González et al., 2017; Anastasiadou et al., 2018; Koch protocols and lower reagent and sample consumption, which will et al., 2018; Gai and Sun, 2019). Recent studies indicate that greatly reduce the cost and the time-to-result. In this context, there exist collaborative activities between these gene regulating label-free optical biosensing devices offer a full range of attractive pathways that results in a common outcome (Murr, 2010; alternatives due to their high-sensitivities and label-free detection Garcia-Gomez et al., 2018; Hanly et al., 2018). On the other approaches, which can be further exploited thanks to their hand, NA molecules from bacteria and viruses are commonly versatility and capabilities for multiplex detection. In particular, employed as markers indicative of the presence of pathogenic evanescent-wave biosensors have achieved great progress for NA microorganisms in infectious disease diagnosis (Fournier et al., analyses (Carrascosa et al., 2016). They have been benefited 2013). However, rather than simply attempting to identify from improvements in biosensor fabrication and production foreign NA, more insight may be gained by investigating the quality (Fernández Gavela et al., 2016; Soler et al., 2019), the different genetic/epigenetic mechanisms which can be affected availability of new surface chemistry methods (Escorihuela et al., by the infection, potentially improving diagnosis by more 2015; Escorihuela and Zuilhof, 2017; Bañuls et al., 2019), the precisely determining infection status and progression or drug availability of highly efficient probes for NA detection (Shi susceptibility (Bierne et al., 2012; Ciarlo et al., 2013; Kathirvel et al., 2015; Nafa et al., 2016; Aviñó et al., 2019a), and new and Mahadevan, 2016). In addition to clinical diagnostics, approaches for the enhancement of the detected signal (Guo understanding the dynamics of these networks could positively et al., 2015). Also, the biosensor integration with microfluidics help and transform current medicine by the development of new permits the incorporation of different modules, including fluidic therapies where it may be possible to revert the altered cellular transportation, sorting, mixing or separation methods for liquid process back to its normal state. For example, novel treatments samples, and the automation of the complete analysis, which pave based on targeted therapy are being developed for melanoma the way for the full development of the so-called lab-on-a-chip patients that harbor a BRAF mutation (Spagnolo et al., 2015). (LoC) platforms (Jung et al., 2015; Szydzik et al., 2015, 2017). Frontiers in Chemistry | www.frontiersin.org 2 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors The objective of this work is to give a comprehensive extends beyond the surface into the external medium, as a so- overview of the analytical strategies that employ evanescent- called evanescent field (Figure 1B). Variations in the refractive wave optical biosensors to deal with the complexities and index of the external medium just above the surface will challenges of effective NA detection, with applications ranging cause a change of the optical properties of the excited optical from identification of SPNs and epigenetic alterations in cancer, mode through this evanescent field, resulting in a variation to the detection of indirect modifications of NA processes caused of its effective refractive index and allowing for a quantitative by bacterial infections. We also identify the main challenges measurement. Due to the exponential decay of the evanescent faced by this optical biosensor technology and describe several field in the external medium (usually few tens to few hundred innovative approaches in order to manage the existing challenges nanometres), only changes occurring in close proximity to the limiting the performance of the current methods. surface will be sensed, with a natural filtering of background from the surrounding medium. Evanescent detection does not become weaker with reduced sample volume, instead it is proportional EVANESCENT-WAVE OPTICAL to the concentration of analyte that come into contact with BIOSENSORS the functionalised surface. This feature makes evanescent wave biosensors particularly attractive for use with reduced sample A biosensor is a self-contained device capable of providing volumes and consequently reduced auxiliary reagent volumes specific quantitative or semi-quantitative analytical information as well. using a selective biological or biomimetic recognition element Evanescent-wave biosensors enable monitoring of (biological receptor) which is in direct spatial contact with a biomolecular interactions in real-time opening the possibility physical transducer (Soler et al., 2019) (Figure 1A). Optical of evaluating the affinity and kinetics of the interaction and can transducers are one of the most commonly used for NA detection contribute to a more concrete disease diagnosis. These type of (Bora, 2013). They detect biological interactions by evaluating biosensors also benefit from versatility—theoretically, it would be the variations induced in the light properties, such as intensity, possible to selectively sense an almost limitless range of analytes wavelength, refractive index, or polarization (Sang et al., 2016). just by selecting the appropriate biological receptor. Currently, They can be classified in label-based or label-free sensors. Label- new options for sensor transducers are emerging due to the based usually detect changes in color or the presence of photons progress in nanofabrication technology which further provide generated at a particular wavelength by optical labels, such as interesting opportunities for miniaturization, high-throughput dyes/DNA intercalators or fluorescent molecules. The label serves and low-cost production (Sagadevan and Periasamy, 2014; Sang as an indirect indicator of the presence of a concrete analyte. et al., 2016). Plasmonic and silicon photonics based biosensors Hybridization between a DNA probe and its specific target has are among the most employed evanescent-wave biosensors been detected by doping with chromophore molecules (Szukalski for the analyses of NAs with potential applicability in clinical et al., 2019). Fluorescence emission from molecular beacons (Yao diagnosis (Ermini et al., 2014; González-Guerrero et al., 2016). et al., 2003), quantum dots (Chen et al., 2010a), or fluorescent- These biosensors have shown high detection sensitivities with labeled signal probes (Zhu et al., 2017) have also been employed short response times. In this review, we will identify the main for NA detection. Although the use of optical labels is widespread challenges faced with plasmonic and silicon photonic biosensors in NA analyses, these methods are inherently time-consuming and summarize some of the most recent analytical strategies and can greatly increase the costs of the assays (Chen et al., developed for the analysis of NA biomarkers with these devices. 2017). In addition, they are prone to sample losses in the labeling process and, in the case of fluorescence, fluorophores are quite sensitive to environment conditions, such as pH, and can be Plasmonic-Based Biosensors bleached very fast, reducing the efficiency of the analysis (Sang Plasmonic-based biosensors constitute the most employed et al., 2016). evanescent-wave optical biosensors for NA analyses. Among On the other hand, label-free optical biosensors enable them, Surface Plasmon Resonance (SPR) biosensor is the measurement of the physical properties of the analytes under most advanced and mature technology (Soler et al., 2019). a label-free scheme, removing experimental ambiguity, and SPR biosensors employ propagating surface plasmons that allowing for more reliable analysis that require minimal assay oscillate collectively on a planar metal-dielectric interface. The development (Chen et al., 2017). There is a vast repertoire of surface plasmon electromagnetic field propagation is constrained label-free optical biosensors able to transform a specific biological vertically at this interface but is free to propagate laterally interaction into a quantifiable signal based on different working along the film. The plasmon wave propagates with very specific principles, including the scattering of light or the generation phase and attenuation properties which depend strongly on of an evanescent wave. Evanescent-wave biosensors have been the dielectric environment. The plasmon mode has a strong recognized as ideal candidates for NA evaluation without the evanescent field just above the surface of the metal facilitating need of any molecular marker (Carrascosa et al., 2016). They biosensing. For the plasmon excitation, a light source should be exploit the ability to confine an electromagnetic field within or effectively coupled to a thin metal layer, which is usually a 45– at the surface of dielectric and/or metal structures in the form 50 nm thick film of gold. There are several configurations for of localized or propagating electromagnetic modes with well- light coupling in SPR sensors to promote the excitation of the defined optical properties. Part of the electromagnetic mode surface plasmons, including prism coupling, waveguide coupling, Frontiers in Chemistry | www.frontiersin.org 3 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors 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. and grating coupling (Soler et al., 2019). Among these, the prism- conditions. Currently, there are already many different SPR based Krestschmann configuration harnessing Attenuated Total systems commercially available which are broadly deployed in Reflection (ATR) is the most widely employed (Figure 1C). In industry, hospitals, or academia (Prabowo et al., 2018). However, this type of configuration, the surface plasmon is manifested they still involve bulk components and specialized procedures, by a drop in the intensity of the light reflected, that strongly increasing their prices and complexity rendering them not depends on the refractive index of the dielectric. This surface affordable for clinical setting. The new trends aim at reducing plasmon can be tracked to study any molecular interaction the components’ costs, including novel light source technology taking place at the gold sensor surface by looking at changes in and detectors by harnessing current technology that can be easily the coupling angle of incidence or in the coupling wavelength. found everywhere, such as smartphones, to decrease the costs and Moreover, in SPR imaging (SPRi) systems, a multiplexed array foster the portability of such biosensor devices (Liu et al., 2015c). format can be achieved by fixing both, the wavelength or the Parallelly, in the last decade, with the progress of angle of the incident light, and measuring the intensity of nanotechnology, a new generation of plasmonic sensors has different spots using a CCD camera (Wong and Olivo, 2014). been introduced that can improve miniaturization, multiplexing SPR biosensors offer high sensitivities ranged between 10−5 and capabilities, and biosensor chip integration. These biosensors 10−6 refractive index units (RIU), corresponding to NA detection use more sophisticated nanostructured metal films or so-called limit within the pM-nM range with optimal biofunctionalization nanoplasmonic structures with features on the scale of the Frontiers in Chemistry | www.frontiersin.org 4 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors wavelength of the incident light or even smaller. In these have been implemented for biosensing, among which optical nanoplasmonic structures, the surface plasmon can be confined resonators and interferometers have been extensively employed in all three dimensions inducing the non-propagating collective for the analysis of NA biomarkers (Carrascosa et al., 2016). oscillation of the metal free electrons termed localized surface The most common optical resonator in the recent literature plasmon resonance (LSPR). This confinement provokes an is the micro-ring resonator. The main advantage of micro- increment in the electromagnetic field near the structures that ring resonator-based biosensors reside in their compactness, the rapidly falls off with distance and minimize LSPR biosensors ease of design and robust fabrication (Ciminelli et al., 2019). susceptibility to variations produced in the external media by, for In a conventional ring resonator arrangement, the light passes example, changes in temperature, and enhance their sensitivity through a straight waveguide which is coupled to a closed- toward small analytes found at very small concentrations. loop waveguide (micro-rings) (Fernández Gavela et al., 2016) Sensitivity can be enhanced by an appropriate design of the (Figure 1E). The surface of the ring structure is uncovered, nanostructures, which can be exploited to promote different generating an evanescent field. In a ring resonator, the sensitivity resonance modes. In fact, these resonances can be modulated will increase depending on the number of round-trips supported to show highly reproducible strong nearfield enhancement and by the micro-ring, which is defined by the quality factor Q. sub-wavelength light confinement that enable to detect specific The biosensor intensity builds-up by multiple interactions with molecular fingerprints (Dong et al., 2017). The simplicity of the the external environment and has reported sensitivities up to coupling of the light has fostered different detection approaches 10−6 –10−7 RIU. (Figure 1D). Also, the facile device miniaturization provides In interferometers, light is generally divided in two light better opportunities for multiplexed device formats, highlighting waves, one employed as a reference and the other one for sensing. the superiority of nanoplasmonic biosensors over conventional Only the sensing-light wave will be susceptible to variations SPR ones. Furthermore, the performance of the nanostructures of the refractive index, which can be detected at the sensor during the detection of NAs can be predicted by the employment output. Technologies based on light interferometry appear to of an universal model by taking into account the optical and be the most sensitive platforms reported. One of the most mass transport aspects, encouraging the development of devices common configuration employed for biosensing is the Mach- with maximized capabilities (Špačková et al., 2018). Zehnder Interferometer (MZI) (Zinoviev et al., 2008). In its basic configuration, MZI has an input single-mode waveguide with Silicon Photonic Based Biosensors power equally split between two arms by a Y-junction. One of the Silicon Photonics biosensors are gaining momentum in the two arms is exposed to the external medium to be sensed, while diagnostic field. They consist of waveguides (i.e., structures the other is employed as a reference (Figure 1F). The two arms of conductive or dielectric material used to guide high- are recombined through a second Y-junction into a single output frequency waves, such as electromagnetic waves in the case of waveguide, resulting in an interference pattern with a determined optical waveguides) that confine light in vertical and horizontal number of fringes of a certain amplitude (visibility factor) dimensions but enable the propagation of light in a longitudinal depending on their sensitivity, which has been demonstrated dimension with minimal loss of energy. Although optical to be 10−7 RIU. However, the design and fabrication of the waveguides are normally used in integrated photonic circuits as MZI should be optimized to achieve a symmetric splitting an optical analogy to electrical wires, they can be also employed as and recombination of the light, as well as balanced losses on high sensitive transducers in biosensor devices. These waveguides both the sensing and reference arms. The BiModal Waveguide are highly compact and can be easily miniaturized and patterned (BiMW) interferometer has been proposed as an alternative in complex forms with extremely predictable behaviors. This to this configuration since it avoids light beam splitting and offers the opportunity to generate arrays of sensors within recombination (Zinoviev et al., 2011). In this interferometer, the same photonic chip for multiplexed analysis. Silicon rather than the sensing and reference beams propagating along photonics sensors are fabricated using conventional silicon physically separated arms of the MZI, sensing is achieved by microfabrication techniques, including photolithography and comparing two different supported modes (fundamental and etching processes. This kind of fabrication allows for wafer-level first order light modes) within the same straight waveguide packaging, producing numerous sensors in the same fabrication (Figure 1G). This results into a device with improved tolerance process with accurate precision and reproducibility, reducing to fabrication variations and a smaller footprint, opening the the time and the manufacturing costs (Fernández Gavela et al., possibility to fabricate more sensors in the same area, with 2016). They have good optical properties, high stability with a consequent increase in the reproducibility and reliability of temperature, inertness to many chemicals and solvents, low the sensing evaluations. In the BiMW, the first order mode surface roughness and a versatile chemical functionalization is far more evanescent than the fundamental mode. Hence, (Escorihuela et al., 2014b). In silicon photonic biosensors, the during the biointeraction event, the fundamental mode would light is confined within a waveguide. The propagating light act as the reference wave being only weakly influenced by modes (guided light modes) also exhibit an evanescent field the biointeraction, while the first order mode will be strongly outside the waveguide surface. This portion of the evanescent influenced by the refractive index changes occurring within the field of the guided light modes is sensitive to refractive index evanescent field. The interference of both modes produces a changes at the sensor surface, which will produce a variation of phase variation signal similar to the MZI, which is collected at the phase velocity of the propagating mode. Several architectures the end of the waveguide. This configuration has demonstrated Frontiers in Chemistry | www.frontiersin.org 5 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors a bulk sensitivity of 10−8 RIU, becoming one of the most The nature of the NA is another aspect that should be sensitive interferometers described in the literature for label- considered. RNA molecules are easily degradable by RNases. free direct detection of NAs (Huertas et al., 2016b, 2017). Their half-life is constrained by the presence of these enzymes Silicon photonic biosensors based on MZI and BiMW are totally and the detection methodology may require the inclusion of integrated devices that include the light beam splitting and purification steps (Baratchi et al., 2014). In addition, target recombination, providing further stabilization to mechanical lengths could also complicate the detection process. While short vibrations (González-Guerrero et al., 2016). However, the sequences may produce lesser sensor signal increments, thereby, interferometric signal has a sinusoidal trend and can lead to worse limit of detection (LOD), long sequences are more prone ambiguity during signal interpretation. These drawbacks been to generate secondary structures by self-loop formation by base overcome at the expense of post-processing of the read-out paring, and the target sequence may be hindered. In the case of signal with the use of different innovative forms of modulation DNA biomarkers, they usually are long and doubled stranded based on all-optical phase modulation (Dante et al., 2012) or sequences, being difficult to capture. In this case, detection optical frequency combs (Knoerzer et al., 2019), improving the approaches may need to include the previous use of an enzyme performance of these integrated photonic biosensors. restriction protocol, denaturation steps or special probe designs Further advances in the complete integration of the optical that enable double strand DNA (ds-DNA) capture. components into the same silicon chip is being pursued in The study of gene regulation pathways implies both, the order to satisfy the necessity of point-of-care systems for their detection and quantification of the NA molecules that participate use in clinical settings or for healthcare diagnosis applications. in such processes, i.e., DNA fragments, mRNA isoforms Auxiliary equipment such as signal acquisition devices or high generated by alternative splicing or micro-RNA regulators, current power supplies occupy significant volume of space, which and the recognition of the epigenetic markers such as the 5- detracts from the benefits provided by the micro/nano scale of methylcytosines in DNA. In addition, although independent these devices. Efforts are directed to the integration of multiple analysis of each type of NA biomarker are proven to enhance functions, such as on-chip lasers. Different materials, either alone the sensitivity and specificity of the assays in comparison or in hybrid approaches, have been employed for the integration other circulating biomarkers (Bratman et al., 2015), multiplexed of lasers in silicon chips, such as germanium (Wirths et al., biomarker detection will definitely help to further understand the 2015; Margetis et al., 2018), or group III/V materials (Roelkens dynamics of a concrete disease and increase the sensitivity of the et al., 2015). While the former increases the power supply of diagnosis (Sullivan et al., 2019). the biosensors, the latter maximize the density of integration Finally, sample pre-treatment and manipulation are critical and, therefore, the portability (Luan et al., 2018). Because they for NA biosensing. Direct evaluation of biofluids such as blood are compatible with complementary metal oxide semiconductor or urine remains challenging and is yet to be effectively solved (CMOS) fabrication, different detectors based on these type of for direct label-free evaluation (Soler et al., 2019). The high materials have been also proposed (Luan et al., 2018). amount of proteins and lipids present in blood can adsorb onto the sensor surface and usually lead to high background signals, obscuring the specific signal of the target and hindering Challenges in Label-Free Optical their recognition. Therefore, the biosensor should be provided Biosensing of Nucleic Acids with a low-fouling biosurface in order to prevent non-specific Sensitivity is one of the main challenges in the detection of NA. adsorptions. In addition, NAs are present not only in circulation, Not only are these biomarkers present in low concentrations in but also inside cells, exosomes, or organelles. In order to avoid real samples, but in most cases, they are related to subtle changes cross-contamination, samples may require the separation of in gene expression. In addition, the dynamic range of recognition specific cells, RNAs, DNAs, etc. (Gai and Sun, 2019). Circulating usually should cover a wide range of NA concentrations, such as DNA needs better plasma separation methods because current the case of miRNA analysis, where the over-expression or sub- protocols include centrifugation steps that are too aggressive, expression usually cover from nanomolar (nM) concentrations promoting circulating cell disruption and, therefore, the release to attomolar (aM) (D’Agata and Spoto, 2019). The outstanding of undesired NA material. In addition, this extraction should sensitivity required for NA analysis can be obtained by the be performed right after sample extraction to prevent from any combination of highly sensitive biosensor transducers and contamination of DNA from circulating blood cells. Here, the signal pre- and/or post-amplification approaches by PCR- integration of the optical biosensor with microfluidic lab-on-a- based methods or signal enhancers, such as nanomaterials or chip (LoC) platforms could provide less aggressive, fast and more DNA/RNA binding proteins. However, this sensitivity not only efficient solutions. depends on the physical transduction mechanism of these devices but is vastly dependent on the biofunctionalization approach and the quality of the selected biorecognition element (González- NUCLEIC-ACID BASED LABEL-FREE Guerrero et al., 2016). The bioreceptor layer must interact with OPTICAL BIOSENSORS sufficient specificity and selectivity with the target NA. It is highly dependent on both, the proper covalent attachment to the In label-free optical biosensors, the biorecognition layer at the surface, and the hybridization event. Successful sensing must also surface of the transducer is of paramount importance. The final ensure discrimination between homologous sequences that could sensitivity and specificity of a biosensor are directly related to the require differentiation between a single nucleotide mismatch. activity of the immobilized molecules and the accessibility of the Frontiers in Chemistry | www.frontiersin.org 6 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors specific targets. In the development of NA biosensors there are (Ermini et al., 2011). An important challenge is the presence two key steps: (i) the biorecognition element design, responsible of regions that can assume conformations by self-hybridization of the system selectivity; and (ii) the surface functionalization and may hide the binding sequence of interest. To avoid self- chemistry, which should ensure: a good surface coverage; proper hybridization, probe length and C-G content are determinant accessibility of the target to the immobilized biolayer; low factors. Probes containing between 15 and 25 bases permit non-specific binding; and good reproducibility and robustness strong hybridization while avoiding self-complementarities and (Bañuls et al., 2013). reducing the likelihood of cross-hybridization from undesired molecules (Ermini et al., 2011). At the same time, a 40– 60% content of C-G bases promotes a stronger hybridization Biorecognition Elements in Nucleic due to higher contribution of stacking interactions during Acid-Based Biosensors hybridization, hence contributing to the stability of the formed The biorecognition element must ensure high selectivity to hybrid (Hormeño et al., 2011). However, excessive CG content enable single-nucleotide resolution, homologous sequences may lead to non-specific hybridization of other sequences discrimination, and negligible cross-hybridization from non- bearing also a high quantity of these nucleotides. specific NA molecules present in the same sample. In NA In some cases, the design of the probes is restricted to biosensors, the conventional biorecognition element is a single- a limited sequence such as the case of short NAs. This stranded DNA (DNA probe) with a specific sequence of difficulty becomes even more challenging due to their high nucleotides of around 9–50 bases, which hybridizes to a heterogeneity, since such sequences have isoform or homologous complementary NA molecule. Whereas, antibody production sequences with differences up to the single mismatch. In these involves their induced expression by the injection of a specific situations, the probe design is constrained, putting at risk the antigen into laboratory or farm animals/cells and their eventual sensitivity and selectivity of the biosensor. Therefore, alternative recovery (Chon and Zarbis-Papastoitsis, 2011), DNA is an easily strategies should be considered. Certain buffer compositions have synthesizable biorecognition element. Current biotechnological traditionally solved cross-hybridization problems. The stability methods permit the in-vitro production of synthetic NAs of NA duplexes can be also compromised by the ionic strength with the desired sequence in large amounts and with high of the solution employed for the analyses (Tan and Chen, 2006). degree of purity (Hughes and Ellington, 2017). They can Structural integrity of DNA has been found to be dependent on be customized depending on their application by introducing the DNA affinity for monovalent cations such as K+ and Na+ different modifications in both the 5’ and the 3’ ends. Thus, (Kielar et al., 2018). Thus, buffer cation content can be fine-tuned structural end-modifications can be introduced in the DNA to obtain an appropriate selectivity. In addition, several agents probe sequence for their direct immobilization over different can be employed in the hybridization buffer to reduce the melting types of inorganic materials to generate functional surfaces temperature (i.e., the temperature corresponding to the midpoint for NA detection at a very low manufacturing cost. In the in the transition from helix to random coil) of the hybridization, design of ss-DNA probes, three factors must be considered: without requiring any temperature control. Formamide is a (i) the functional group that will allow the attachment of denaturant compound that has been shown to reduce the melting the probe to the sensor surface; (ii) a vertical spacer to temperature in aqueous solutions, increasing the hybridization improve accessibility, and (iii) the sequence itself (Figure 2A). efficiency (Fontenete et al., 2016; Huertas et al., 2016a, 2017). A wide variety of functional groups are available for synthetic Tetramethylamonium chloride (TMAC) and glycine betaine have oligonucleotides depending on the surface chemistry selected been reported to be isostabilizing agents, altering the melting for the attachment. Short oligonucleotides modified by amino, temperature and making the hybridization solely dependent on thiol, hydrazide, phosphorothioates, or biotin are commonly oligonucleotide length, despite their GC content (Duby et al., used for DNA immobilization (Zourob, 2010). End modification 2004; Napolitano et al., 2004; Schwinefus et al., 2007). The of DNA probes not only introduces a site-specific group for destabilizing effect of such agents increases with increasing GC their oriented covalent attachment, but also allows insertion content, with almost no effect on poly(dAdT) (Schwinefus et al., of a spacer between the probes and the surface. This vertical 2013). On the other hand, some other components, such as urea, spacer improves the mobility of the immobilized probes and have the opposite effect, promoting a decrease in the strength of their accessibility by the complementary target sequences. They AT/U bonds more than GC base pairs (Schwinefus et al., 2007). also move the DNA sequence away from the sensor surface, These agents can be employed alone or in combination in order reducing the adsorption and steric effects (Carrascosa et al., to achieve the proper hybridization efficiency depending on the 2012). Different vertical spacers can be introduced, such as a nucleotide sequence of the targets (Huertas et al., 2016b). chain of 6 or 12 carbons (C6 or C12, respectively) (Schmieder Enhanced target capture efficiency, and therefore, contribute et al., 2016) or poly-thymine (polyTm ) sequences of different to increase the sensitivity of these biosensors, can also be achieved lengths (Huertas et al., 2017, 2018) which acts as a vertical spacer by introducing nucleotides exhibiting restricted conformation due to the low affinity of thymine bases for gold surfaces (Opdahl that promote the base stacking and backbone pre-organization, et al., 2007). such as nucleic acids (LNA) (Fontenete et al., 2016). The LNA For the selection of the probe sequence there are available is a ribonucleotide homolog with a characteristic 2’-O,4’-C- many commercially manufactured and well-understood codes methylene bridge that can increase the melting temperature by that help to tailor the probe-target stability of a given application 2–8◦ C per subunit introduced into DNA or RNA oligomer. Frontiers in Chemistry | www.frontiersin.org 7 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors 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. This configuration provides higher binding affinity and foster 2018). Functional nucleic acids (FNAs) such as aptamers have discrimination of base mismatches, as well as minimize the also been developed using a combinatorial method called SELEX risk of digestion by nucleases (Bakthavathsalam et al., 2018). (systematic evolution of ligands by exponential enrichment), Peptide nucleic acids (PNAs) are DNA/RNA analogs where making possible to evolve NAs in test tubes to bind to a diverse the sugar-phosphate backbone is substituted by units of N-2- range of analytes beyond DNA or RNA with high affinity and aminoethylglycine (Gupta et al., 2017). Their backbone is neutral specificity (Liu et al., 2009). Further structural modifications of and generates no electrostatic repulsions, allowing a remarkable these aptamers have been proven to outperform the aptamer stability toward complementary NAs. They are also able to affinity (Aviñó et al., 2019b). However, biosensor applications interact with both, DNA (Kirillova et al., 2015) and RNA (Sato using this type of biorecognition elements are out of the scope et al., 2016) duplexes and PNAs derivatives can be obtained of this review. Table 1 summarizes the existing types of probes by chemical modification for enhanced affinity (Annoni et al., used in NA biosensors. 2016) and solubility (Sahu et al., 2011). Phosphorodiamidate morpholino oligos (PMO) are also synthetic DNA analogs that Surface Functionalization for Probe possess a neutral backbone of morpholine rings, which not only Immobilization and Antifouling Coating provides higher solubility in aqueous solutions than PNAs, but Physical adsorption is the simplest immobilization method. also more flexibility in length (Liao et al., 2017). Probes based This approach does not require any NA modification taking on triplex-affinity capture are gaining much interest for the advantage of intermolecular forces such as electrostatic, detection of NAs in biosensing platforms (Aviñó et al., 2019a). hydrophobic, and/or polar interactions (Zourob, 2010). NA triplexes can be induced by the interaction of DNA or However, this adsorption is solely governed by physical attractive RNA molecules with a hairpin-like polypurine-polypyrimidine- forces between the biomolecule and the sensor surface, so that rich sequence. They have proven to increase the hybridization the amount of adsorbed DNA probes cannot be controlled and efficiency of long and highly structured RNAs (Carrascosa et al., may vary along the surface, representing an important drawback 2012) and small RNA sequences (Aviñó et al., 2016). They for the biosensor performance. In addition, flow-through assays also show high potential for interacting with duplex DNA or changes in the pH or buffer composition can easily lead fragments by promoting strand displacement (Huertas et al., to desorption of the biomolecules (Rabe et al., 2011), causing Frontiers in Chemistry | www.frontiersin.org 8 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors TABLE 1 | Different types of probes, main characteristics, target sequences, and surface properties with certain small-molecules that can act as advantages. both, lateral spacers and blocking agents, such as 6-mercapto-1- Probe Main characteristics Target Advantages hexanol (MCH) (Satjapipat et al., 2001; Lee et al., 2006b), long alkanethiols (Gong et al., 2006), or thiolated oligoethileneglycols DNA Conventional sequence Single-stranded Simple synthesis (Lee et al., 2006a). These molecules also promote the orientation NAs of the probe, reduce their density and improve the resistance LNA 2’-O,4’-C-methylene Single-stranded Increase melting Tª, affinity to non-specific adsorptions (Satjapipat et al., 2001; Gong et al., bridge NAs and mismatch 2006). The adsorption of poly-adenine (polyA) sequences to the discrimination Decrease nuclease digestion gold surface has been proposed as an alternative to SH-DNA probes (Sohreiner et al., 2010). This approach relies on the PNA N-2-aminoethylglycine Single and Increase hybrids complex backbone (neutral charge) Double-strand stability unusually strong interaction between adenine nucleotides NAs and gold. PMO Morpholine rings backbone Double-strand Increase solubility and The surface of an SPR sensor can also be modified with NAs length flexibility a functional layer that carries various functional end-groups, DNA Polypurine/polypyrimidine- Double-strand Increase hybridization such as maleimide (Lee et al., 2007), amine (Brockman et al., clamp rich sequences NAs and efficiency 1999), or carboxyl (Burgener et al., 2000) groups for the Antiparallel sequences structured NAs subsequent covalent immobilization of the probes (Figure 2C). 8-aminoG modifications Carboxyl (-CO2 H) groups are widely employed, and usually involves the covalent bonding of amine-modified DNA (NH2 - DNA) probes. This covalent coupling consists of an amide a loss of the signal and possible cross-contamination of the bond formed between the primary -NH2 of the DNA probe surface. On the other hand, covalent immobilization approaches and the -CO2 H terminated monolayer, which is previously involve chemical reactions to link the biomolecules to the activated by the well-known carbodiimide-mediated chemistry. sensor surface by covalent bonds. Covalent linking generally A solution containing a mix of EDC/NHS (1-Ethyl-3-(3- requires the modification of the oligonucleotide probe during dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide) is the synthesis process with a functional group to guarantee employed to activate the -CO2 H groups by producing a NHS- the appropriate chemical bonding. Chemical grafting prevents ester intermediate highly reactive to the primary amine in the the release of probes into the solution, surpassing one of the DNA probe (Conde et al., 2014). main drawbacks of physical adsorption and promoting better Other strategies make use of affinity linkers, such as sensor reproducibility. Likewise, covalent linking can reduce biotinylated probes, which can be efficiently immobilized on the background signal coming from non-specific adsorption. avidin/streptavidin modified surfaces (Vaisocherová et al., 2006; Different functionalization strategies for gold and silicon surfaces Mir et al., 2008; Biagetti et al., 2018). The interaction between for the development of label-free optical NA biosensors will be avidin/streptavidin with biotin is known to be strongest protein- introduced in the next sections. ligand non-covalent bond. Once formed, it is very rapid and remains stable even under extremes pHs and temperatures, DNA Immobilization Strategies for Gold Surfaces when dissolved in organic solutions, or in the presence of other Gold sensor surfaces are the most employed in plasmonic denaturing agents. biosensors. Chemical adsorption (i.e., chemisorption) of thiolated molecules on gold is the most widely used DNA Immobilization Strategies for Silicon-Based immobilization approach due to its easy preparation. This Surfaces approach takes advantage of the strong affinity of thiol atoms In silicon-based NA biosensors, surface modification with toward gold surfaces (Sakao et al., 2005). Chemisorption of DNA organofunctional alkoxysilanes has received widespread probes modified with a thiol linker, i.e., SH-DNA probes, is application due to their considerable stability and rapid covalent the most straightforward approach for the development of NA linkage (Hu et al., 2015). Although silane SAM formation biosensors (Figure 2C), generating self-assembled monolayers in silicon surfaces is not as straightforward as thiol SAM on gold (SAMs) carrying directly the probe sequence that will hybridize surfaces, it shows higher physical and chemical stability, since it with the target. The grafting density of SH-DNA probes is allows to implement a wide variety of chemical reactions (Bañuls high compared to those obtained using other immobilization et al., 2013). Silane SAMs are obtained by a chemisorption of methods (Peeters et al., 2008) and it is affected by the length of trichloro-, trimethoxy-, or triethoxy molecules, onto the sensor the immobilized probes (Ulman, 1996), pH (Xia et al., 2010), surface followed by condensation of this molecules with hydroxyl or the salt content of the employed buffer (Satjapipat et al., groups generated at the silicon surface. The silanization reaction 2001). However, a highly dense and compact DNA monolayer will be strongly influenced by the experimental conditions, is not always ideal, since it can hinder the accessibility of target where the most relevant parameters are the nature of the sequences due to steric and electrostatic forces, diminishing silane, its concentration, nature of the solvent, water content, the likelihood of hybridization and, therefore, the detection temperature, and time (Bañuls et al., 2013). Silanes can have signal (Lee et al., 2006b) (Figure 2B). Hybridization signal different functional groups which introduce the specific surface can be enhanced by optimization of the DNA coverage and functionalization for the subsequent immobilization of the Frontiers in Chemistry | www.frontiersin.org 9 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors probes (Figure 2D). Amino-terminated silanes, such as APTES to other biomolecules (e.g., proteins, lipids, etc.), further (3-aminopropyltriethoxysilane), is one of the most widely used hindering their specific detection in real samples. Therefore, the for biofunctionalization due to their reactivity with different development of biosensor strategies for the analysis of complex functionalities, such as aldehyde, carboxylic acid, and epoxy. solutions directly without the need for any purification steps APTES silane can incorporate different crosslinkers, a class of is on demand. Several reviews have been published describing molecules capable of linking two functional groups together, different low-fouling coatings for label-free optical biosensors e.g., surface groups and biorecognition elements, in solution. (Liu et al., 2016; Vaisocherová-Lísalová et al., 2016). BS3 (bis(sulfosuccinimidyl)suberate), a water-soluble amine- A common approach to reduce the fouling effects is to-amine homobifunctional crosslinker, can be employed for the addition of different surfactants (e.g., Tween), protein- the covalent bonding of NH2 -DNA probes (Cardenosa-Rubio based additives (including BSA or casein), or non-protein et al., 2018). This crosslinker contains an NHS ester at each end reagents. These approaches minimize any hydrophobic that enable the formation of amide bonds when reacting with and/or electrostatic attractions between the complex matrix primary amines. Amino-silane surfaces can be also activated and the functionalised surfaces. Unfortunately, they have with 1,4-phenylenediisothiocyanate (PDITC), which provide limited antifouling properties (Vaisocherová et al., 2009). isocyanate groups that can be reactive either to NH-DNA Functionalization methods should enable the immobilization of probes (Hu et al., 2015), yielding thiourea bonds, or to SH- biorecognition elements and include components that provide DNA probes (Huertas et al., 2016b), forming thiocarbamate a low-fouling background in complex solution. Hydrophilic bonds. However, the most used homobifunctional crosslinker surfaces are particularly amenable for NA hybridization because is glutaraldehyde, which forms a surface with aldehyde groups they facilitate exposure of hybridizing bases (Chen et al., 2010b). that lets the formation of imines by their covalent reaction Low-fouling surfaces based on poly(ethylene glycol) (PEG) and with amine groups (Zainuddin et al., 2018). The fact that its derivatives have been used widely to solve the problems homobifunctional crosslinkers carry two identical chemical arisen in the analyses of complex solutions (Blättler et al., 2006; groups can cause undesired side effects such crosslinking Soler et al., 2014). PEG molecules create a brushed coating between surface groups or biorecognition elements, which can on the surface, which has proven to successfully prevent and actually inhibit the functionalization process. In this regard, reduce non-specific adsorption of proteins due to its hydrophilic heterobifunctional crosslinkers can overcome those side-effects properties (Soler et al., 2014). In addition, recent advances due to the different nature of their reactive groups (Jin et al., in polymer chemistry have led to the development of various 2003). Other silanes carry SH- as the functional groups, such as polymer coatings (e.g., zwitterionic and non-ionic polymer MPTS (Mercaptopropyltriethoxysilane). Thiolated monolayers brushes) with high resistance to fouling from complex biological allow rapid and simple immobilization of SH-DNA probes by fluids (Rodriguez-Emmenegger et al., 2011). different chemistries without using any crosslinker or other reagent, such as disulphide bond linkage (Sánchez del Rio et al., Amplification Strategies 2007), or through thiol-ene click photochemistry (Escorihuela Amplification strategies facilitate the identification and et al., 2014a). The latter one can also be also employed by quantification of extremely low-concentrated samples or activation of the thiol group at the probe to further reaction small targets which do not have enough mass, size, and/or with different functional groups at the sensor surface, such concentration to generate a significant change in the refractive as alkenylated and alkynylated surfaces by forming thiol-ene index when they are analyzed with a label-free optical biosensor. and thiol-yne links, respectively (Bañuls et al., 2019). These These approaches are very convenient when the sensitivity of approaches can be enhanced by the use of polythiolated the transducer does not range the required physiological levels. probes, generating multiple anchor points at the sensor surface Several amplification strategies have been reviewed for optical (Bañuls et al., 2017). biosensing elsewhere (Zhou et al., 2018a,b). Here we summarize some of the most employed strategies (Figure 3). Generation of Antifouling Monolayers The performance of label-free optical biosensors may be Enzyme-Based Amplification compromised by interfering effects that produce refractive This amplification approach makes use of an enzyme (most index changes unrelated to the analyte binding, referred to often DNA polymerase) to create a large number of copies as fouling effects (Vaisocherová-Lísalová et al., 2016). When of a specific NA. The most widely employed technique in the target molecules are contained in a complex solution, biology is the well-known polymerase chain reaction (PCR). the specific response due to their capture may be concealed To amplify the analyte, it is necessary to increase the number due to the adsorption of non-target molecules. These fouling of copies of the target sequence, increasing its concentration, effects can be particularly significant during the analysis of prior to the interaction with the biosensor. PCR can be very complex clinical samples such as blood plasma, serum, or effective, however it presents a number of challenges, like the urine. There are many non-specific interactions between the requirement for sophisticated technology, for example thermal surface and the complex matrix components such as hydrogen cyclers, which delay the detection times (Martzy et al., 2019). bonds, hydrophobic, electrostatic, and/or polar interactions Isothermal PCR is a PCR-based strategy that is carried out at a making the reduction of the background signal a difficult task. constant temperature, increasing the speed of analysis, avoiding Circulating NAs are present in very low concentrations compared alternating temperature cycles. It enables higher amplification Frontiers in Chemistry | www.frontiersin.org 10 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors 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. efficiencies in less amount of time compared to traditional PCR developed a biosensor to identify miR-29a which is a biomarker (Yoon et al., 2015). Asymmetric PCR (aPCR) preferentially of influenza infection. amplifies one strand of the original DNA over the other, allowing selective generation of single stranded DNA (ss-DNA) Self-Catalytic Amplification amplicons by introducing one of the PCR primers at a much There exist methods based on enzyme-free amplification which larger concentration during amplification (Graybill et al., 2018). can achieve augment the copy number without enzymes or These products will easily hybridize with conventional duplex amplification labels, using protocols that are simpler, more DNA probes by nucleotide complementarity without requiring stable and lower-cost. Hybridization chain reaction (HCR) is any DNA denature step. These different approaches are usually a self-catalytic amplification strategy which initiates when the performed prior to hybridization of the target to the probe target hybridizes with a complementary hairpin capture probe at the sensor surface. However, other amplification methods (Figure 3B). When the stem-loop structure of the capture probe act once the probe is attached to the sensor surface and use is unfolded, it creates a one-dimensional DNA structure, and it as the initiator of the amplification process. The rolling two partially complementary DNA hairpins couple with it and circle amplification (RCA) strategy employs padlock probes trigger the hybridization reaction, producing copolymers which (PLPs), which are single-stranded NA probes that consist of amplify the sensor signal (Dirks and Pierce, 2004). This process ′ ′ three main parts: the 5 and 3 ends that hybridize with the can be enhanced by the use of a hairpin-free system in which ds- target sequence, a sequence that interacts with the capture DNA monomers assembled into a dendritic nanostructure when probes, and a universal primer binding site that generate it interacts with a trigger sequence which initiates the non-linear repeated sequences. Once the target sequence is recognized, HCR (Ding et al., 2017). it is enzymatically joined by DNA ligase forming a circular Another approach consisting of Catalyzed Hairpin Assembly probe-target complex. This is then amplified isothermally, (CHA) has achieved an SPR signal amplification during miRNA generating concatenates of ss-DNA with multiple repeats of analysis (Li et al., 2016a). Recognition of miRNA target off-chip the complementary sequence (Dean et al., 2001). Xiang et al. by a hairpin (H1) triggers the interaction of the latter with a (2015) identify Mycobacterium genomic DNA with a detection second hairpin (H2). This interaction releases the miRNA target limit of 10 pM by employing a SPR biosensor and AuNPs for cyclic reuse and CHA products are generated creating more for amplification. than 100-fold amplification. The main drawback from traditional Nucleases can be seized for signal amplification allowing CHA is the background signal generated by the non-specific recycling of the target due to the digestion of the probe. These CHA products when the target is not present. To overcome this enzymes can hydrolyse the phosphate diester bond between limitation, mismatched base pairs can be introduced into the nucleotides, degrading NAs (Figure 3A). Ki et al. (2019) and Wei breathing sites of the hairpins (Li et al., 2016b). et al. (2018) used nuclease to free a DNA initiator that generate DNA sandwiches, leading a LoD for miRNA detection of 2.45 Protein Binding Nucleic Acids and 0.15 picoMolar (pM), respectively. In a similar way, RNase One of most commonly employed amplification strategies is H can be used to degrade miRNA capture probes to recycle the use of a specific antibody directed to DNA-RNA hybrids the cDNA target, amplifying the sensor signal. Ho et al. (2017) (i.e., anti-DNA-RNA antibody) (Boguslawski et al., 1986). The Frontiers in Chemistry | www.frontiersin.org 11 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors antibody will bind to the DNA-RNA duplexes leading to a usually fabricated using soft-lithography methods (Luka et al., signal proportional to the concentration of the hybridized 2015). They are mostly fabricated in polymer material such RNA (Figure 3C). This allows for the quantification of RNA as polyethylmethacrylate (PMMA) and polydimethylsiloxane concentrations out of the range of the detection limit of (PDMS) due to their excellent chemical, physical, and mechanical the biosensor. The main advantage of the antibody is that properties, and excellent biocompatibility. Microfluidics have it recognizes hybrids regardless of the sequence, allowing been harness for label-free NA detection, and is a very promising multiplexed measurements (Schmieder et al., 2016; Sguassero platform for parallelization of analyses, increasing the flow- et al., 2019). This approach is of interest for the detection of throughput of the sample while minimizing sample volumes, short RNA sequences, which can be difficult to amplify through which significantly reduces the cost of the assays (Baratchi PCR or related methods. Also, it shows very rapid time-to-result et al., 2014). Microfluidics has enabled the label-free detection of and may enable three orders of magnitude improvement of the different NAs such as mRNAs (Huertas et al., 2017) or miRNAs detection limits. (Graybill et al., 2018) in multiplexed devices by the creation of independent parallel channels that allow sample delivery over Nanomaterials specific biosensors. The high molecular weight of nanomaterials can be exploited In addition, different microfluidic designs can be fabricated to increase the refractive index change for each target binding on a single chip and disposed in customized arrangements event by an increment of the mass on the sensor surface. to perform different procedures (Jung et al., 2015). This is Gold nanoparticles (AuNPs) are used frequently for signal of particular interest in NA analyses, since protocols usually amplification in label-free optical biosensors, especially in involve multiple steps, such as sample lysis to expose NAs found plasmonic ones. These nanoparticles are usually functionalised in the cells or exosomes, their purification and extraction or with specific molecules that are able to interact with the their amplification to increase the target copy number before hybridized target at the sensor surface, such as complementary the eventual detection by the biosensors, as discussed in the DNA probes that create super sandwich structures (Figure 3D) previous section. mRNA isolation has been achieved by the (Vaisocherová et al., 2015; Liu et al., 2017; Melaine et al., purification with oligo (dT) that selectively hybridize to the 2017) or anti DNA-RNA hybrid antibody (Sguassero et al., poly (A) tail found mRNAs, achieving up to 70% efficiency 2019). Gold nanoparticles not only increase the mass, but it (Satterfield et al., 2007). The functionalised photopolymerized has also been shown that the local SPR of the particle can lead monolith was employed to purify mRNA from eukaryotic cell to further increase in the apparent effective refractive index total RNA. An RNA isolation microfluidics have been developed due to a phenomenon called coupling effect. The coupling capable of obtaining high-quality RNA from different biological effect is a distance dependent factor, achieving maximum samples within 30 min (Yoon et al., 2018). This device took enhancement within 8 nm distance of the particle from the advantage of the capability of dimethyl adipimidate agent to surface for 20 nm diameter AuNPs. Outside this distance, bind to RNA in a pH-dependent and reversible manner, avoiding the size/mass properties are the major factors in the signal the employment of chaotropic salts or spare solvent. The non- improvement (Hong and Hall, 2012). Similar properties chaotropic nature of this molecule allowed to simplify the have been demonstrated with gold nanorods, enabling SPR isolation process and highly reduced any contamination derived signal enhancement for the detection of miRNAs at the from the conventional methods, enhancing the quality of the femtomolar level (Hao et al., 2017). In addition, graphene extracted RNA (≈87% recovery rate). Similarly, non-chaotropic sheets can be enriched with AuNPs, forming graphene agents can be applied for DNA extraction and the potential oxide-AuNPs composites (GO-AuNPs) that can offer the of this methodology has been demonstrated for the analysis of physicochemical properties of both materials. Additionally, genetic and epigenetic alterations (Shin et al., 2014), leading to GO-AuNPs can be used in two ways: as sensor substrate, high quality and quantity of DNA purification from urine and providing a high surface area to significantly increase the blood samples. The high quality of the extracted material enables immobilized bioreceptors; and as an amplification element further on-chip amplification through PCR-based methods to (Li et al., 2017). Gold nanoparticle-decorated molybdenum increase the sensor sensitivity. Haber et al. (2017) developed sulfide (AuNPs-MoS2 ) can also be created by enriching the a microfluidic platform for qPCR integrated with an LSPR edge or defective sites of MoS2 sheets with gold nanoparticles biosensor. The microfluidic PCR chip utilized piezoelectrically- (Nie et al., 2017). pumped recirculating flow. They demonstrated multiplexed detection of E. coli DNA target amplification within 15 min, achieving a LoD of 5 fg/µL. Also, an integrated microfluidic PCR MICROFLUIDICS SYSTEMS FOR NA device and an SPR-fiber sensor has allowed DNA amplification ANALYSIS of Salmonella spp. and its downstream label-free detection (Nguyen et al., 2017). In this case, a serpentine micro-channel The unprecedent development of label-free optical biosensors was fabricated over two copper heat blocks, allowing on-chip due to technological evolution from macro, to micro,- and thermal cycling. nanotechnologies has been further enhanced by integration with These findings highlight the potential of combining label- microfluidic circuits. Conventional microfluidic devices rely on free optical biosensors with different microfluidic modules the continuous flow of liquids in channels of a few microns for a faster and more user-friendly operation. In addition, Frontiers in Chemistry | www.frontiersin.org 12 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors automated microfluidics for NA analyses can be achieved by respectively. They also evaluated the hybridization rates from incorporating a series of microvalves and micropumps (Kim a mix of perfect/mismatched sequences with different ratios. et al., 2012). These automated circuits have been proven to be The non-specific interactions of the mismatched sequence with of considerable value when integrated to multiplexed photonic the capture probe suggested that the development of different chips (Szydzik et al., 2017), presenting considerable advantages bioreceptors, such as PNA or new bioreceptors could improve the in the fabrication of LoC platforms (Jung et al., 2015). All in all, specificity and the selectivity. Indeed, a new neutralized chimeric microfluidic integration empowers label-free optical biosensors DNA analog bioreceptor was developed to detect perfect matched with exceptional capabilities and contribute to the development sequences, discriminating mismatched ones (Huang et al., of portable and user-friendly devices which can be used at the 2018). The chimeric DNA probes contained four methylated doctor’s office or the patient’s home (González-Guerrero et al., nucleotides in the backbone for charge neutralization and a NH2 2016; Lopez et al., 2017). group in the 5’-end for its immobilization in a glutaraldehyde- activated SAM. By using an SPRi biosensor, they evaluated the efficiency of this new bioreceptor to discriminate sequences APPLICATIONS FOR CLINICAL DIAGNOSIS that differed in a single nucleotide, demonstrating a favorable DNA Punctual Mutations hybridization of perfectly matched DNA sequences with their SNPs are produced by the change of a single nucleotide for chimeric probe under specific conditions of temperature and another one within a gene sequence. This means that the ionic strength. The activity of this chimeric DNA could be translation process from mRNA to protein might suffer some potentially enhanced by carefully designing probes sequences alteration resulting in no translation of a protein, incorrect with different methyl sites. folding or translation of a non-functional protein. The percentage Jin et al. (2017) developed a micro-ring resonator sensor of SPNs in human samples is typically very low and the specificity combined with isothermal solid-phase amplification/detection required should have resolutions at the single nucleotide level. (ISAD) to identify G12D and G13D mutations found in codon Therefore, in the last years, the efforts have been focused in 12 and codon 13 of KRAS gene from 70 colon rectal cancer tissue increasing the sensitivity and specificity of SNP analysis through samples (Figure 4A). The sensor surface was functionalized with different label-free optical biosensor approaches in a faster and G12D or G13D mutant primers followed by an on-chip ISAD. less laborious manner (Table 2). Nguyen and Sim (2015) used an Since only the mutant primers were immobilized, mutant alleles LSPR based on scattered Rayleigh light from gold nanoparticles were specifically amplified from the clinical samples while wild- to identify E545K and E542K mutations in PIK3CA gene from type ones displayed no sensor signal. The ISAD-KRAS approach ctDNA. They biofunctionalized gold nanoparticles with a specific showed more specificity and selectivity than PCR and direct PNA to promote the specificity at the single mismatch level, sequencing methods, detecting samples containing only 1% of the which is an important requisite for NA detection. These PNA mutant allele compared to the 30% shown by these techniques. hybridized 100% with ctDNA and mismatched with normal By evaluating 70 cases of colorectal cancer, ISAD-KRAS detected circulant DNA, achieving a limit of detection of 200 fM. In even cases where PCR and direct sequencing did not have addition, this methodology also enabled recognition of the DNA sufficient sensitivity. A similar approach was followed by Liu methylation profile of the captured sequences by immunogold et al. (2015b) to identify punctual mutations in the EGFR gene, colloid functionalised with anti-5-methylcytosine (anti-5-mC) specifically, the L858R mutation. They identified 1% of mutant antibody, obtaining an enhanced LSPR resonant signal only from alleles in a sample of mixed cellular types in contrast with the 25% the methylated DNA due to the presence of the immunogold achieved by PCR. Moreover, they evaluated lung cancer patient colloids. Through this secondary detection approach, they were samples, correctly detecting the L858R mutation in samples that able to identify 2 mCpG sites on the ctDNA and improve the were also detected by direct sequencing. It was also possible detection limit by a factor of four. Another LSPR biosensor to identify cases that the PCR could not detect due to the was developed to compare the hybridization rates of perfectly low amount of DNA. Both ISAD-based approaches have been matched and mismatched sequences that only differed in one demonstrated to be promising diagnosis tools for SNPs analyses nucleotide in codon 12 of KRAS gene (Rapisarda et al., 2017). In and appealing alternatives to PCR and sequencing methods due this study, SH-DNA probes were employed for a direct and label- to their fast operation time (<30 min), sensitivity, specificity, and free detection of the sequences, achieving LoD of 10 and 13 nM, cost-effective relation. TABLE 2 | Optical biosensors for DNA punctual mutations detection: mutations, genes involved, real samples analyzed, and limit of detection achieved. Sensor SNP Sample LoD Amplification References LSPR E545K and E542K (PIK3CA) ctDNA in plasma 200 fM Immunogold colloids Nguyen and Sim, 2015 50 fM LSPR codon 12 of (KRAS) Buffer 10 nM and 13 nM NA Rapisarda et al., 2017 Micro-ring resonators G12D and G13D in KRAS Colorectal cancer tissue 1% mutant allele ISAD Jin et al., 2017 MZI L858R in EGFR Lung cancer tissue 1% mutant alleles ISAD Liu et al., 2015b Frontiers in Chemistry | www.frontiersin.org 13 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors 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. DNA Methylation Profile of MCF-7 cells. Methylated sequences produced a shift in the DNA methylation is an epigenetic mark found in some cytosines resonant wavelength of the resonator immobilized with methyl- generally located in CpG islands of most promoter regions. The specific primers, whereas no amplification was observed in the level of methylation of these regions determines the accessibility resonator functionalised with the unmethyl-specific ones. The of the transcription factor to the promoter and, therefore, the described LoC enabled identification of methylation levels of levels of expression of the genes. Normally, the hypermethylation 1% in a mix of methylated/unmethylated DNA compared to of these CpGs regions are related to down-transcription and, the 10% achieved by RT-PCR. It also eliminated the human inversely, hypomethylation means overexpression (Rodriguez errors introduced during the manual bisulfite treatment and et al., 2006). The detection of the methylation status using could identify the DNA methylation status in only 80 min of label-free optical biosensors has attracted increasing attention analysis, in contrast with conventional methods (24 h). However, over the last few years (Nazmul Islam et al., 2017). As per it did not suppress the long and intense processes related to the SNPs, the concentration of these biomarkers is rather low bisulfite conversion. In addition, bisulfite conversion cannot be in samples (Gai and Sun, 2019) (Table 3). In addition, they used to identify other epigenetic marks generated by enzymatic should be able to specifically detect the methylation status oxidization and that are highly important for the assessment at the single cytosine resolution for more accurate analyses. of the methylation process, such as of 5-mC, such as 5- Yoon et al. (2015) developed a LoC device based on micro- hydroxymethylcytosines (5-hmC), 5-formylcytosine (5-faC), and ring resonators and bisulfite conversion methodology. Bisulfite 5-carboxylcytosine (5-caC) (Chowdhury et al., 2014). Due to this is a carcinogenic compound which converts the non-methylated lack of versatility, other detection approaches have been pursued. cytosines into uracil whereas methylated cytosines remain intact. Methyl-cytosines and their derivatives can be specifically A pre-processing module including a microchamber for bisulfite analyzed by the employment of specific antibodies and proteins. treatment, a micromixer that passively mixed the sample, and Kurita et al. (2015) designed a microfluidic device to measure a DNA-purification microchannel were integrated on the same DNA methylation in an SPR sensor. They mixed fragmented chip and coupled to a detection module. After the purification DNA with a biotinylated bulge inducer probe, exposing the step, an ISAD was performed using methyl- and unmethyl- cytosine in a looped-out conformation. Then, this DNA-bulge specific primers grafted in micro-ring resonators for the analysis complex was attached on a streptavidin surface by streptavidin- of the methylation profile of RARβ gene from genomic DNA biotin interaction. Finally, they identified the methylated Frontiers in Chemistry | www.frontiersin.org 14 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors TABLE 3 | Optical biosensors for DNA methylation profiles identification: genes involved, real samples analyzed, methodology employed, and detection limit reached. Sensor Gene Sample Methodology LOD Amplification References SPR Synthesized ODN genomic λ DNA and HCT116 biotinylated bulge inducer probe/ 1 cytosine in 48 fg NA Kurita et al., 2015 human colon cancer cells anti-5-mC SPR Synthesized PAX-5 Buffer poly-purine reverse-Hoogsten Single-cytosine NA Huertas et al., 2018 gene hairpin/anti-5-mC SPR MGMT promoter gene genomic λ DNA alkylating linker 25% NA Kurinomaru et al., 2017 molecule/anti-5-mC LSPR p53 gene promoter DNA extraction of HeLa and PNA/methyl-CpG binding Single-cytosine NA Nguyen et al., 2015 HEK 293 cells domain protein 2 Micro-ring RARβ gene genomic DNA of MCF-7 cells Methylated/unmethylated 1% in methylated/ ISAD Yoon et al., 2015 resonators primers unmethylated mixture Toroidal resonant Synthesized ODN Buffer Anti-5-hmC 0.42 pM NA Hawk and Armani, cavity 2015 cytosine using an anti-5-mC antibody, achieving single-cytosine hairpin (PPRH) probes that permitted the direct recognition resolution in 48 fg of genomic HCT116 human colon cancer of ds-DNA by inducing strand displacement and promoting cells. The simplicity and miniaturization of the microfluidic triple-helix formation (Figure 4B). The PPRH probe presented sensor offer great advantages for this methodology. Nevertheless, 8-aminoG modifications to enhance the strength of the capture, a denaturalization step off-chip is necessary to allow the target which further increased the stabilization of the triple helix hybridization with the bulge inducer probe, which leads to structure. The efficient capture of ds-DNA fragments allowed to an increase in the detection time. Different approaches have quantify and assess different methylation profiles (from 1 to 4 been developed in order to directly capture intact ds-DNA cytosines) in PAX-5 gene promoter through immunodetection targets without the need for denaturalization steps, resolving using anti-5-mC antibody. the problem of the direct analysis of ds-DNA molecules. DNA methylation analysis based on immunodetection allows DNA methylation status has been analyzed by the direct for the investigation of other epigenetic modifications rather immobilization of ds-DNA on gold surfaces by employing an than 5-mC, which can be detected by employing the specific alkylating linker molecule, L1 (Kurinomaru et al., 2017). This antibodies for each one. Hawk and Armani (2015) employed molecule consists of two reactive groups, nitrogen mustards, a toroidal resonant cavity sensor for the identification of DNA that interacts with DNA, and cyclic disulphides, attaching containing 5-hydroxymethylcytosines by using a specific anti- to the gold substrate. Using an SPR biosensor, they directly 5-hmC antibody. In this case, instead of using a DNA probe, immobilized methylated ds-DNA on the gold sensor surface and they attached the antibody to the sensor surface. Hence, direct assessed the methylation levels employing anti-5-mC antibody. detection of synthetized ds-DNA with methylated, unmethylated This approach could be a promising tool to decipher global and hydroxymethylated epigenetics modifications was carried methylation status but lacks specificity for concrete DNA out, showing a limit of detection of 0.42 pM and demonstrating methylation sequences. Much of hybridization-based analytical the versatility of the assay for different epigenetic modifications. techniques struggle to capture ds-DNA due to the use of probes which analytical performances are limited to duplex Long RNAs formation. Alternative approaches have been implemented to Long RNAs are characterized by a length from 200 bases up to overcome such limitation. Nguyen et al. (2015) employed an several kilobases. They can be classified as coding RNAs and non- LSPR biosensor based on gold nanostars and Rayleigh resonance coding RNAs depending on if they encode the information (i.e., to assess the methylation profile of p53 gene promoter. They mRNA) for the synthesis of proteins or not. The latter ones have biofunctionalized the gold surface with a specific PNA, which structural [transfer RNA (tRNA), ribosomal RNA (rRNA), small enables to form stable complexes with ds-DNA. They quantified nuclear RNA (snRNA)] and regulatory roles [Long intergenic the methylation status of the captured ds-DNA target by specific non-coding RNAs (lncRNAs); Natural Antisense Transcripts; interaction with methyl-CpG binding domain protein 2. This Promoter associated ncRNAs, etc.] (Carrascosa et al., 2016). One biosensor achieved a limit of detection down to the single- advantage of long-RNA biosensing is the large refractive index cytosine and it was applied to DNA samples from HeLa and changes they produce due to their large mases, which is translated HEK 293 cells-extraction. Moreover, it allowed the study of to higher sensor signal intensities. Table 4 summarizes different conformational changes of p53 gene promoter and the relative strategies for long RNA biosensing. transcriptional efficiencies regulated by methylation, highlighting its potential as a tool to study the effects generated by the presence Messenger RNAs of this epigenetic mark. Huertas et al. (2018) also achieved a mRNA analysis gives information about the translation of a direct detection of ds-DNA and the evaluation of methylation particular protein, allowing even the identification of the cell status employing an SPR biosensor within 30 min. They used types that are suffering changes in the translation process a novel bioreceptor based on poly-purine reverse-Hoogsten that might be related to the beginning of some forms of Frontiers in Chemistry | www.frontiersin.org 15 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors TABLE 4 | Optical biosensors for long RNAs detection: type of RNA, real samples analyzed, strategies to improve the sensitivity, and the demonstrated limit of detection. Sensor RNA Sample LOD Amplification References SPR Fas57/Fas567 mRNA alternative HeLa cell total RNA 387/438 pM NA Huertas et al., 2016a splicing SPRi 16S rRNAs L. pneumophila, P.auriginosa and 10 pg/mL AuNPs Melaine et al., 2017 S.typhimurium total RNA Micro-ring lncRNAs KIAA0495/PDAM and Patient-derived xenograft GBM6 RNA C(t) values aPCR Cardenosa-Rubio et al., resonator MALAT1 2018 BiMW Fas57/Fas567 mRNA alternative Buffer 580/735 fM NA Huertas et al., 2017 splicing diseases. Discrimination among the diverse mRNAs transcripts at the sensor surface. Subsequently, they performed a signal is essential. Alternative splicing process can generate different amplification based on Au-NP functionalized with a DNA probe mRNA isoforms from a common pre-mRNA. These isoforms specific to a second target region of the previously captured share common exon sequences that might result in cross- rRNA sequences. The amplification approach resulted in a 3- hybridization. For all these reasons, a small number of label- fold increase of the SPR signal, achieving a limit of detection free optical biosensors have been developed for monitoring of 10 pg/mL and a range of detection of 0.01–100 ng/mL, which alternative splicing processes for diagnostic purposes. Huertas covers the wide dynamic range of recognition necessary for the et al. (2016a) developed an SPR biosensor for the direct detection of the NA expression dynamics as highlighted in the quantification of Fas gene alternative splicing mRNA isoforms challenges. Total RNA from the different bacterial cultures were (Fas57/Fas567, Figure 5A). Prior to detection, they optimized analyzed after RNA extraction and fragmentation by a multiplex a fragmentation step based on RNA alkaline hydrolysis to approach that allowed to detect in a selective and fast way 16S customize the isoforms’ length to the biosensor convenience rRNA from the three different bacterial strains. (≈200-bases fragments). This strategy allowed to overcome the challenge encountered in the detection of long RNA molecules Long Non-coding RNAs and open the door to explore new biomarkers in evanescent- The first lncRNA discovered and studied was H19 (Huang et al., wave optical biosensors based on long RNA targets. For detection, 2019). Since then, many additional lncRNAs have been identified they immobilized SH-DNA probes on the gold sensor surface and investigated as they interfere in several biological functions. containing the splice-junction sequences, the key feature for For example, KIAA0495/PDAM can act as tumor suppressors the specific recognition of each isoform. Also, they optimized in oligodendrioglioma while MALAT1 (metastasis associated a highly stringent buffer by adjusting with the formamide lung adenocarcinoma transcript 1) is related to different content and the ionic strength, achieving 100% selectivity and tumors being able to act as tumor suppressor in glioblastoma. a limit of detection of 387/438 pM for each isoform. The Cardenosa-Rubio et al. (2018) used a micro-ring resonator for methodology was validated by evaluating purified RNA samples the multiplexed detection of the three lncRNAs (Figure 5C). from different HeLa cell lineages, which were in good agreement They carried out a PCR amplification prior detection, allowing with conventional RT-qPCR-based methodology. In addition, the subsequent hybridization of the generated ss-DNAs with further implementation in a multiplexed BiMW biosensor complementary DNA probes spotted in array distribution in outperformed the SPR biosensor, exhibiting LODs with three the silicon sensor surface. The selected lncRNAs were tested in order of magnitude improvement thanks to the high sensitivity spiked-in commercial brain and lung total RNA samples and in of these interferometric biosensors (Huertas et al., 2017). RNA from glioblastoma cell line (GBM6). Results were validated by RT-qPCR and showed high consistency with results from Ribosomal RNAs precedent literature. Ribosomes are hybrid complexes formed by proteins and rRNA sequences that forms part of a large ribosomal subunit Short Non-coding RNAs and a small one. Although the different subunits have highly Small RNAs are characterized by a short length around 20–200 conserved molecular weights and sequences, they differ among bases and they englobe microRNAs (21–25 bases), piwiRNAs organisms, enabling creation of phylogenies and identification (20–30 bases), tinyRNAs (<22 bases) and small interfering RNAs of the microorganisms. rRNA is present in high concentrations (20–25 bases). All play a structural or regulatory role so they are inside the cell, but its secondary structure may hinder the called small non-coding RNAs because they are not translated recognition event by DNA probes, limiting the sensitivity. into proteins (Carrascosa et al., 2016). Most label-free optical Melaine et al. (2017) used a SPRi sensor for the simultaneous biosensors have been harnessed for the detection of miRNAs detection of 16S rRNAs (a component of the small ribosomal (Table 5). Schmieder et al. (2016) employed an SPR biosensor subunit in procariotes) from three pathogenic bacterial strains: to detect miRNA-93. To maximize both, the selectivity and L. pneumophila, P. aeruginosa, and S. typhimurium (Figure 5B). sensitivity of the SPR detection, they used a SH-LNA probe and They selectively captured the targets by duplex DNA probes subsequent amplification by anti-DNA/RNA hybrid antibody, Frontiers in Chemistry | www.frontiersin.org 16 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors 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. enhancing the sensor response by one order of magnitude. producing a strong change in mass with a correspondingly large Enhanced amplification can be achieved by the combination of signal. This method was capable of simultaneously detecting this antibody with Au-NPs. Sguassero et al. (2019) introduced miR-21 and miR-192 at the low picomolar range in 10% diluted a multiplexed SPRi biosensor for the detection of four miRNAs bovine serum. An analogous strategy was developed by Ki et al. potentially involved in multiple sclerosis. The antibody permitted (2019) (Figure 6A). They used an LSPR biosensor based on the recognition of the different miRNA sequences selectively gold nanostars to detect miR-10b in urine and plasma samples hybridized on the sensor surface, producing an enhanced SPRi from mice with orthotopic Hs746tT xenografts. Amplification signal correspondent to the concentration of captured miRNAs. was performed by duplex-specific nuclease that degraded the This approach was used to analyse to RNA extracted from DNA probe in DNA/RNA hybrids, recycling the target and blood samples from multiple sclerosis patients and succeeded in liberating a DNA initiator. DNA initiator was captured creating detection of relevant miRNAs at the sub-picomolar range. a DNA sandwich with DNA helpers and tannic acid gold The versatility of label-free optical biosensors allows nanotags, allowing 2.42 pM LoD. Results showed that miR-10b integration of different amplification methods within the same concentration was higher in plasma than in urine samples. assay. Wei et al. (2018) employed an SPRi biosensor for the Dual amplification was achieved by the generation of two multiplexed assessment of miR-21 and miR-192 in buffer. layers of GO-AuNPs composite in an SPR sensor (Li et al., 2017). In order to increase the sensitivity, they integrated strand The bottom layer served as a surface for the immobilization of displacement amplification (SDA) with gold nanoparticles. SH-DNA probes on the nanoparticles, providing functionality to To do so, they designed hairpin probes for the amplification the sensor. In a second step, the target miRNA was hybridized that consisted of three different domains, including the with the immobilized probes and subsequently interacted with miRNA-recognition sequence, an amplification domain and a DNA functionalized GO-AuNPs composites, forming the upper common recognition domain which hybridizes with both, a layer that acted as a enhancer of the sensor signal. They showed SH-DNA probe immobilized at the sensor surface and the gold one order of magnitude LOD improvement in the two-layer nanoparticles. The different miRNAs interacted specifically with approach compared to the performance of the single layer their recognition sequence in each amplification probe, causing used as a substrate, achieving extremely sensitive detection (0.1 the opening of the hairpin probe loop and initiating the SDA. fM) of miRNA-141 extracted from different cancer cell lines. Finally, the released triggers hybridized with the captured probes In addition, they obtained corresponding levels of expression on the sensor surface and the DNA functionalized AuNPs, with RT-qPCR. A similar strategy was developed based on gold Frontiers in Chemistry | www.frontiersin.org 17 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors TABLE 5 | Optical biosensors for miRNA detection: strategies to improve the sensitivity, the demonstrated limit of detection and the real samples analyzed. Sensor Target miRNA Sample LOD Amplification References SPR miR-145 Buffer 1 nM NA Aviñó et al., 2016 SPR miR-93 Buffer 10 pM Antibody Schmieder et al., 2016 SPR miR-141 total RNA extracted from cancer cell lines 0.1 fM GO-AuNPs Li et al., 2017 (prostate: 22Rv1; hepatocellular:SMMC7721; colon LoVo) SPR miR-21 Hepatocarcinoma cell line (Bel-7404); 0.6 fM AuNPs Liu et al., 2017 SMMC-7721; L-02; Hela SPR miR-141 total RNA extracted from cancer cell lines 0.5 fM AuNPs-MoS2 Nie et al., 2017 (prostate: 22Rv1; hepatocellular: SMMC7721; colon LoVo); 10 % human serum SPR miR-21 total RNA extracted from MCF-7 cells 1 pM CHA Li et al., 2016b SPR miR-21 total RNA extracted from MCF-7 cells < 1 pM CHA Li et al., 2016a SPRi miR-21 and Buffer 0.15 pM (miR-21) and 0.22 Nuclease + AuNPs Wei et al., 2018 miR-192 pM (miR192) SPRi miR-422; total RNA extracted from blood 0.5 pM AuNP + antibody Sguassero et al., 2019 miR-223; miR-126; miR-23a SPRi miR-29-3p total RNA extracted from throat swab RNase H Ho et al., 2017 LSPR miR-10b urine and plasma samples of mice with 2.45 pM Nuclease + HCR + tannic tags Ki et al., 2019 orthotopic Hs746tT xenografts Mirroring miRNA-let7f, RNA extracted surgical glioma cells Ct aPCR Graybill et al., 2018 resonator miRNA-219, miRNA-10b, miRNA-29a, miRNA-335, miRNA-124a, miRNA-222, miRNA-34a, miRNA-155 MZI miR-21, let-7a urine (bladder cancer) and cell lines (MCF7 1 nM NA Liu et al., 2015a and A549) BiMW miR-181a Urine (bladder cancer) 23 aM NA Huertas et al., 2016b nanoparticles-decorated molybdenum sulfide (AuNPs-MoS2) They identified two miRNAs (miR-21 and let-7a) that participate as amplification element (Nie et al., 2017). SH-DNA probes in bladder cancer onset in human urine samples and obtained a complementary to miR-141 were immobilized on an SPR LOD of 1 fmol/µL (1 nM). In addition, they identified SNPs from biosensor to identify miR-141 in the same cellular lines and HeLa let-7 family miRNAs. Finally, attomolar detection limit of miR- cells, achieving a limit of detection of 0.5 fM. Moreover, they 181 (LoD = 23 aM) in a direct assay (<20 min) was achieved assessed the accuracy in spike samples diluted in 10% human using a BiMW interferometer (Huertas et al., 2016b) (Figure 6B). serum acquiring reliable values validated by RT-qPCR. The ultra-high sensitivity achieved reveals the potential of these Other methods to increase the sensitivity during miRNA types of optical transducers for the analysis of extremely low detection are based on probe design. Aviñó et al. (2016) concentrations with no need for any amplification strategy. synthetized a new DNA tail-clamp purine track to detect miR- They discriminated homologous miRNAs at single nucleotide 145. This probe promoted a triple helix with the RNA target, mismatched, as well as pre-miRNA-181, during the evaluation improving 2.4 times the sensitivity over duplex forming probes. of miR-181 in urine samples from cancer bladder patients. ′ In addition, an 8-Amino-2 -deoxyguanosine modification was These results uncovered the participation of this miRNA in the introduced to stabilize the triplex by 1.5 times, making this new development of bladder cancer. probe attractive for improved miRNA detection. Graybill et al. (2018) employed a micro-ring resonator array to analyse eight miRNAs from surgical glioma cells. They amplified CONCLUSIONS AND FUTURE the signal using aPCR and evaluated the hybridization rate of PERSPECTIVES ss-DNA as Ct, like qPCR, achieving values of level expression similar to the literature for surgical glioma cells. Also, a MZI The employment of evanescent-wave optical biosensors for was employed for the detection of miRNAs by Liu et al. (2015a). the routine analysis of NA-based biomarkers threatens to Frontiers in Chemistry | www.frontiersin.org 18 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors 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. change the concept of diagnosis. The opportunity for fast, new microfluidic approaches should be implemented to meet highly sensitive, and multiplexed analyses of the genetic and the specific pre- and post-processing requirements for each epigenetic landscape of different diseases makes these devices biomarker inside the same sensor platform guaranteeing and a very attractive diagnostic solution. Numerous biosensing testing their stability and reproducibility. In addition, sample approaches have been proposed as solutions for use in clinical volumes should be reduced to few microliters. environments. Thanks to their highly sensitive transducers On the other hand, the vast genetic and epigenetic network and their ease to be combined with different amplification makes calls for a new system for well-defined biomarker panels methods, label-free optical biosensors have allowed NA detection linked to concrete diseases and disease stages. Extensive testing in a wide dynamic range of concentrations, from nanomolar in clinical trials is required to establish standard patterns for each down to attomolar. Their versatility to incorporate specialized case. Future trends should be focused on leveraging integrated probes and immunodetection methods have also enhanced microfluidics and multiplexed label-free optical biosensors to the selectivity of the analysis for the identification of a great create highly complex diagnostic, monitoring and prognostic variety of NA biomarkers, including SNPs, methylation patterns, systems. These complex systems will unlock even more detailed mRNAs, and miRNAs. In addition, these sensors are well-suited and specific information. The sheer vastness and subtlety of to integration within lab-on-a-chip microsystems, enabling the form and function of the NA biomarkers suggest that big further enhancement through combination with a variety of data and bioinformatic analysis approaches will be required to pre- and post-processes, thanks to monolithic integration extract useful information from these analyses. Such an endeavor with complex and automated microfluidic systems, ultimately will contribute to making connections between specific diseases enabling the development of low-cost, multiplexed, and user- and signatures. Interactivity in the form of potentially reversible friendly platforms. epigenetics will add an additional dimension. Unfortunately, there still exist some drawbacks to be On the whole, the analysis of genetic and epigenetic surpassed. Most of the applications described in this work biomarkers is a key requirement for the effective analysis and are preclinical proof-of-concepts performed in the very understanding of important malignancies, such as infectious controlled laboratory environment environments. This can diseases and cancer. The potential reversibility of genetic and difficult the complete translation of the technology to real epigenetic mechanisms poses great promise in the development clinical settings. Further investigations need to be fulfilled of personalized treatments for each patient. Indeed, in precision to confirm their feasibility which should be aligned with medicine, the aim is the molecular characterization of each internationally established guidelines. Efforts should be focused patient, which can only be realized by the employment of low- on the development of biosensors capable of simultaneous cost and high throughput screening technologies. There is a lot multiplexed analyses in a few minutes. In terms of integration, that can be gleaned on patient-derived data with PoC devices, Frontiers in Chemistry | www.frontiersin.org 19 October 2019 | Volume 7 | Article 724 Huertas et al. Advanced Nucleic Acid Optical Biosensors which can be very valuable. Such a holistic approach can also be of CERCA program/Generalitat de Catalunya. ICN2 was value for personalized drug delivery, effectively provide tailored supported by the Severo Ochoa Centres of Excellence program medications and therapies, ensuring that the most adequate funded by the Spanish Research Agency (AEI, grant no. treatment is given to the right patient with a proper dose at the SEV-2017-0706). ICN2 and the authors acknowledge the correct time. support of the Spanish MINECO, the Spanish Research Agency and the European Social Fund (ESF) for the grant AUTHOR CONTRIBUTIONS for predoctoral contracts in the Subprograma Estatal de Formación del Programa Estatal de Promoción del Talento CH and LL devised the review. CH and OC-L conducted the y su Empleabilidad (ref. BES-2017-080527) linked to the literature review and provided the first draft. CH and OC-L TEC 2016-78515-R project. created the figures. CH, OC-L, AM, and LL contributed to manuscript revision and approved the submitted version. ACKNOWLEDGMENTS FUNDING A substantial part of the content of this review has been adapted from the doctoral thesis Nanophotonics biosensors for deciphering CH was a recipient of an RMIT Vice Chancellor’s cell regulation pathways written by the corresponding author CH Postdoctoral Fellowship. The ICN2 was funded by the and LL (Huertas et al., 2016c). REFERENCES Belic, J., Koch, M., Ulz, P., Auer, M., Gerhalter, T., Mohan, S., et al. (2016). Mfast-seqs as a monitoring and pre-screening tool for tumor-specific Anastasiadou, E., Jacob, L. S., and Slack, F. J. (2018). Non-coding RNA networks in aneuploidy in plasma DNA. Adv. Exp. Med. Biol. 924, 147–155. cancer. Nat. Rev. Cancer 18, 5–18. doi: 10.1038/nrc.2017.99 doi: 10.1007/978-3-319-42044-8_28 Annoni, C., Endoh, T., Hnedzko, D., Rozners, E., and Sugimoto, N. (2016). Bellassai, N., and Spoto, G. (2016). 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