This article is made available via the ACS COVID-19 subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. pubs.acs.org/acssensors Review How Nanophotonic Label-Free Biosensors Can Contribute to Rapid and Massive Diagnostics of Respiratory Virus Infections: COVID-19 Case Maria Soler,*,‡ Maria Carmen Estevez,‡ Maria Cardenosa-Rubio, Alejandro Astua, and Laura M. Lechuga Cite This: ACS Sens. 2020, 5, 2663−2678 Read Online ACCESS See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Downloaded via UNIV AUTONOMA DE BARCELONA on October 15, 2020 at 12:41:27 (UTC). Metrics & More Article Recommendations ABSTRACT: The global sanitary crisis caused by the emergence of the respiratory virus SARS-CoV-2 and the COVID-19 outbreak has revealed the urgent need for rapid, accurate, and affordable diagnostic tests to broadly and massively monitor the population in order to properly manage and control the spread of the pandemic. Current diagnostic techniques essentially rely on polymerase chain reaction (PCR) tests, which provide the required sensitivity and specificity. However, its relatively long time-to-result, including sample transport to a specialized laboratory, delays massive detection. Rapid lateral flow tests (both antigen and serological tests) are a remarkable alternative for rapid point-of-care diagnostics, but they exhibit critical limitations as they do not always achieve the required sensitivity for reliable diagnostics and surveillance. Next-generation diagnostic tools capable of overcoming all the above limitations are in demand, and optical biosensors are an excellent option to surpass such critical issues. Label-free nanophotonic biosensors offer high sensitivity and operational robustness with an enormous potential for integration in compact autonomous devices to be delivered out-of-the-lab at the point-of-care (POC). Taking the current COVID-19 pandemic as a critical case scenario, we provide an overview of the diagnostic techniques for respiratory viruses and analyze how nanophotonic biosensors can contribute to improving such diagnostics. We review the ongoing published work using this biosensor technology for intact virus detection, nucleic acid detection or serological tests, and the key factors for bringing nanophotonic POC biosensors to accurate and effective COVID-19 diagnosis on the short term. KEYWORDS: point-of-care diagnostics, optical biosensors, nanoplasmonics, silicon photonics, virus detection, coronavirus, covid-19, respiratory virus, label-free detection V iral respiratory tract infections (RTI) are one of the leading causes of hospitalization in developed countries and a common cause of death.1 In developing countries, the concern is Wuhan (China). In such a short period since the appearance of the first suspicious cases and further confirmation, the virus has massively spread worldwide, affecting all countries and even greater due to the shortfall in healthcare (vaccines, efficient territories, leaving to date (July 2020) more than 14 million diagnostics, clinical resources, etc.), nutrition, and hygienic infected people and nearly 600,000 deaths. The surprisingly fast measures.2 Since the discovery of influenza in the early 1930s, propagation of SARS-CoV-2 has been due to factors such as its tens of common viruses have been identified, and in the past contact and airborne routes of transmission (i.e., transfer from decades, new emerging viruses have appeared with increasing contaminated fomites to mouth, nose, and eyes, and also via frequency, gradually warning the scientific and social commun- aerosols generated and remaining in suspension when sneezing, ity toward improving identification, surveillance, and control to coughing, breathing, and talking), its high infection rate, long prevent and stop them from causing epidemics. After the critical incubation period from exposure to onset of symptoms, outbreaks of SARS (Severe Acute Respiratory Syndrome) in 2003, H1N1 flu (swine flu) in 2009, and MERS (Middle East Respiratory Syndrome) in 2012, it has been in 2019−2020 when Received: June 11, 2020 the world has been globally dealing with the most critical health Accepted: August 7, 2020 situation in years due to the SARS-CoV-2 (a coronavirus closely Published: August 7, 2020 related to MERS-CoV and SARS-CoV) causing the COVID-19 outbreak. COVID-19 has become a global pandemic in less than four months since its appearance in late December 2019 in © 2020 American Chemical Society https://dx.doi.org/10.1021/acssensors.0c01180 2663 ACS Sens. 2020, 5, 2663−2678 ACS Sensors pubs.acs.org/acssensors Review contagious time (three-week period from onset of symptoms), amplification and detection of specific sequences of the viral the variety of symptoms (fatigue, fever, cough, dyspnea···) and, nucleic acids, are currently well established in routine analysis more importantly, the lack of symptomatology, which hinders a and indeed have drastically redefined the diagnostics protocol rapid identification, facilitating its imperceptible spread. The and efficiency. The relatively fast access to the genomic sequence significance of this crisis has also sadly revealed the lack of global of the pathogens, even in newly emerged viruses, such as the readiness from governments, public organizations, and SARS-CoV-2, enables the rapid design and production of highly communities to face and manage both the social and health specific PCR kits for its ready and sensitive detection, and even consequences of the outburst. The scientific community is now offering quantitative values of the viral load (typically between globally challenged to provide solutions in three main areas: (1) 100 and 1000 copies/mL). This has been evidenced with prevention, through the development of efficient vaccines that COVID-19, when within the first months after the outbreak, provide immunity and protection to the population; (2) many PCR kits were commercialized and distributed, offering therapeutics, through the development of specific drugs and excellent sensitivity and specificity, turning them into the gold therapies to treat and cure the disease; and (3) diagnostics, standard diagnostics technique for SARS-CoV-2 infection. through the implementation of massive, affordable, and reliable Although the analysis is relatively fast (i.e., less than 2 h), the detection tests. sample-to-result time can be considerably longer (i.e., from 4−6 Once the emergence has been recognized and identified, it is h to a few days), mainly due to sample transportation and crucial to initiate actions to stop the virus transmission and treatments that must be performed in centralized laboratories. prevent the spread. Besides social distancing and confinement, The initial limitations of PCR techniques related to reduced the strategy must also involve careful tracing and massive testing multiplexing capabilities have also improved significantly. Many of the community, which has dramatically revealed the need for companies offer either detection kits for the three most common efficient, readily accessible diagnostics methods that specifically respiratory viruses (influenza A/B and the respiratory syncytial confirm the presence of the virus as soon as possible. Given the virus, RSV), or more sophisticated platforms which offer health and social emergency, mass-produced, reliable, and very cartridges for the detection of panels of 6, 12, or even 20 sensitive diagnostics to promptly identify the pathogen are then respiratory pathogens.5,6 In all cases, the performance of such mandatory and a paramount priority to improve patient testing platforms renders sensitivity and specificity values management, infection control, and more efficient epidemio- around 95−100% and could be adapted to include the novel logical studies. coronavirus.6 However, most of them are not compatible with In this review, we provide an overview of current and new point-of-care testing, do not offer quantitative information, are diagnostic strategies that can be implemented for SARS-CoV-2 necessarily linked to their commercial instrument, and are detection. We mainly focus on label-free nanophotonic relatively costly.7 biosensors as a potential technology for rapid and efficient Current efforts are mostly directed to point-of-care (POC) virus infection diagnostics. The main working principles and decentralization, to accelerate and promote the analysis outside characteristics of nanophotonic biosensors are briefly described the lab, close to the sample collection,8 and already a couple of together with examples of their recent applications for them have received express authorization to be used for respiratory virus detection. qualitative detection of SARS-CoV-2, such as the Accula test ■ DIAGNOSTIC STRATEGIES FOR RESPIRATORY VIRUS INFECTION from Mesa Biotech Inc. or the ID Now test from Abbott Diagnostics. The development in recent years of new NAAT- based techniques that circumvent the use of thermocyclers, Viral culture has been for a long time the traditional gold required for up and down temperature cycles in PCR, have standard diagnostics technique for RTI, as it provides high boosted these new designs, easing the portability and eventually specificity, moderate sensitivity, and quantitative information. the transfer into POC devices.9,10 This is the case, for instance, However, it is time-consuming (5 to 20 days) and requires of Loop-Mediated Isothermal Amplification (LAMP) or Rolling experienced staff for results interpretation. Current diagnostic Circle Amplification (RCA) techniques, which also rely on DNA methods for respiratory tract infections are divided into three amplification, but are designed to operate at constant temper- distinct approaches:3,4 ature (60−65 or 37 °C, respectively).11−14 Both have been (1) Detection of the viral genome through nucleic acid implemented for the detection of several pathogens, including amplification tests (NAATs), mainly polymerase chain viruses such SARS-CoV,15,16 H5 avian influenza virus,17 and also reaction (PCR) tests in its different variants; SARS-CoV-2.18 Besides, microarray-based methods, enabling multiplexed assays and with a high potential for POC testing, (2) Direct detection and identification of the intact virus (or have been designed and applied for different virus detection fragments) through the recognition of viral antigens (influenza, RSV, generic CoV, MERS-CoV, etc.).19,20 Finally, (structural proteins of the virus); CRISPR-based techniques are also emerging as novel diagnostic (3) Indirect detection of antibodies produced by the infected tools, using the RNA-targeting Cas13 enzyme for highly specific host during the course of the infection, known as pathogen detection, which is being intensively studied and serological testing. applied as well for COVID-19 diagnostics.21−23 Strategies 1 and 2 usually require a nasopharyngeal sample Antigen-Directed Virus Detection. Antigen-directed (nasal swabs), whose viral load is generally high, and they are diagnostics involve the detection of the virus via recognition useful while the infection is active; whereas strategy 3 requires a elements (commonly specific antibodies) that capture exposed blood sample and it can only be applied from day 4 or 5 after the proteins in the structure (viral antigens), such as the S (spike) infection. protein in coronaviruses, or the HA protein (hemagglutinin Nucleic Acid Amplification Tests (NAATs). NAATs based glycoprotein) in influenza virus. Conventional approaches, such on PCR in its different variants (reverse-transcriptase PCR, RT- as the enzyme-linked immunosorbent assays (ELISA) or PCR, real-time quantitative PCR, qPCR, etc.) for the immunofluorescence staining, are used to identify the virus 2664 https://dx.doi.org/10.1021/acssensors.0c01180 ACS Sens. 2020, 5, 2663−2678 ACS Sensors pubs.acs.org/acssensors Review and provide qualitative or semiquantitative results with adequate respiratory viruses, such as adenoviruses, influenza A and B sensitivity. Although very specific, they require highly skilled viruses, parainfluenza, or RSV. These kits can provide more personnel for optimal accuracy, and they are time- and labor- reliable and quantifiable data and are meant to be used in central consuming.3,24−26 A few of them are also commercialized either laboratories with appropriate readers. However, the most for the detection of a panel of respiratory viruses with its straightforward and most widely commercialized assays rely benchtop instrument, such as mariPOC tests (via sandwich on LFA tests, analogous to those for virus rapid detection in test- immunoassay run in automatized mode), or for individual plate- strip format, with the viral antigens being adsorbed on the paper- based kits, although they are not commonly used for routine based pads. Most of them provide information on the type of diagnosis. antibody detected (IgG and/or IgM), which may help Instead, rapid antigen diagnostic tests (RADTs) based on differentiate different stages of the infection (e.g., acute phase immunochromatography (IC) or lateral flow assays (LFA) have or past infection). Due to the emergency situation, COVID-19 expanded widely, adapting the immunoassay concept to serology studies are currently intensively performed worldwide disposable, inexpensive, fast (around 15 to 60 min turnaround for both diagnostics and pandemic monitoring purposes,33−35 time), qualitative (yes/no), and point-of-care testing. The and these tests are under development and commercialization. RADTs are cellulose-membrane strip tests, commonly using a Both microplate format immunoassays (ELISA) and rapid LFA sandwich assay. The sample containing the virus (or viral tests have been the main focus,8,18,36 although not with the antigens) is placed in the sample pad area, which flows by desired outcome in the case of rapid tests. Most of the capillarity to differentiated areas: first, a pad containing specific commercialized tests lack minimum sensitivity and reproduci- labeled (fluorescent tag, gold nanoparticles, etc.) antibodies bility to be reliable and accurate, partially due to the lack of against the antigens, which captures the analyte (if present). rigorous quality and accuracy controls before commercializa- When the sample gets to the detection line, the analyte− tion. Besides, LFA tests only provide qualitative information antibody complex is captured by other immobilized specific (yes/no to the presence of antibodies). As the COVID-19 antibodies, forming a sandwich complex and revealing the disease is not well-known yet, the immune response according to presence of the viral antigen in the sample. These tests are the severity (asymptomatic, mild, or severe outcome) or the commercially available for most common respiratory viruses duration of the acquired immunity, if any, requires a highly such as influenza A and B or the RSV.6 Some RADTs have also reliable, sensitive, and ideally quantitative strategy, which been urgently developed and launched for COVID-19 current LFA-based techniques are not providing. diagnostics.18 For example, Coris BioConcept has launched a Optical Biosensors as Potential Integrated Diagnostic dipstick rapid test (COVID-19 Ag Respi-Strip) with a claimed Approach. For respiratory virus infections, especially of specificity of around 98% but a sensitivity of about 60%; SD emergent viruses, which we are facing more often, a more Biosensor, with the Standard Q COVID-19 Ag test, reaches a integral diagnostic approach that provides fast and quantitative sensitivity of 84%. Despite the advantages for POC massive information is highly needed. Having a unique single platform testing, these tests have critical issues, such as the very limited that can group all three diagnostic strategies discussed above, sensitivity (especially in adults)27 and a poor PPV (positive combining the best features of all of them, represents an unmet predictive value), which is more evidently revealed when they need that currently available technologies cannot provide. are compared with nucleic acid tests, whose levels of accuracy Developing, implementing, and commercializing a device which and sensitivity are very high. The low reliability of these tests can deliver the specificity and accuracy of nucleic acid-based might be in part due to the limited quality of biological material detection, the convenience of fast detection of the virus directly employed for their development (both antibodies and antigens, in the extracted sample, or the detection and quantification of used for either the antibody production or the assay develop- antibody titer directly in collected serum or blood, at the point- ment and validation), which essentially requires time for of-care, with enough reproducibility, accuracy, and sensitivity, is optimization, and is especially manifest with a new, unknown a very challenging task. Optical biosensors are at the forefront in emerging virus. this singular race, as they provide outstanding characteristics as Serological Assays. Serology assays are based on the excellent levels of sensitivity, robustness, and immunity to indirect identification of the virus infection, through the specific electromagnetic interferences, miniaturization and integration detection of the antibodies generated by the infected host during capabilities, and portability, among others.37,38 Therefore, the immune response. Usually, this process starts a few days after optical biosensors are excellent candidates to move the analysis infection, and once the virus has been cleared from the organism, from centralized laboratories to the point-of-care.39,40 Optical the antibodies remain in the bloodstream, generally for several biosensors measure variations of the optical properties of the years (although it depends on the disease and the pathogen). propagated light (i.e., absorption, polarization, intensity, wave- This approach is useful for diagnostics (particularly in length, dispersion, or refractive index) when the interaction retrospective diagnosis), but it is especially attractive for between the receptor (antibody, nucleic acid sequences, surveillance and to perform epidemiological studies,28,29 to enzymes, proteins, etc.) and the target analyte takes place. estimate the prevalence, and to monitor the levels of antibodies Among them, the ones founded on the evanescent field sensing over time, which help assess the duration of the acquired principle, such as the photonic and plasmonic biosensors,41−43 protection or immunity. Antibodies generated by the body often have already demonstrated their enormous potential as sensing target key antigenic determinants in the pathogen, for example, tools in a myriad of environmental, industrial, pharmaceutical, the structural proteins of the virus. Thus, the detection and, especially, clinical scenarios.44−46 Evanescent wave optical mechanism of this strategy involves the analysis of blood (or biosensors operate in a label-free configuration (no fluorescent, serum) from the patient and the antibody detection through colorimetric, enzymatic tag is needed), and their level of their specific capture by those viral antigens.30−32 sensitivity allows them to get rid of further amplification steps There are several enzyme immunoassay (ELISA) kits that prolong and complicate the assay. They have the potential available for the detection of serum antibodies to most common to increase the multiplexing capability, facilitating the 2665 https://dx.doi.org/10.1021/acssensors.0c01180 ACS Sens. 2020, 5, 2663−2678 ACS Sensors pubs.acs.org/acssensors Review simultaneous detection of several targets, by having different previously and can be explained on the basis of the evanescent sensing channels within the same chip. As they rely on the field sensing principle.40,42,49,50 In optics, an evanescent wave is monitoring of changes occurring on the surface of the biosensor formed when light traveling in a medium undergoes total chip, they can also be literally adapted to any kind of target (for internal reflection (TIR), generating a near-field enhancement example, detection of the virus, nucleic acid sequences, or at the boundaries that penetrates in the surrounding dielectric antibodies; see Figure 1), as long as carefully selected receptors medium with a vanishing intensity. The evanescent field is are immobilized on the surface, to ensure the sensitivity and extremely sensitive to changes in the refractive index (RI) of the specificity requirements for a given application. medium, which are translated in more drastic changes of light properties such as the intensity, phase, resonance momentum, or polarization. It is important to note that the intensity of the evanescent field decays exponentially; therefore, the sensing probe is restricted to the immediate vicinity of the transducer surface and expanding up to a few tens of nanometers depending on the transducer. In other words, when a biomolecular interaction or binding occurs at the sensor surface within the evanescent field, it can be directly monitored by interrogating wavelength displacements, light intensity, or phase variations, among others, in a real-time and label-free format with impressive sensitivity. Nanoplasmonic Biosensors. The Surface Plasmon Reso- nance (SPR) system is the landmark of label-free nanophotonic biosensors. The SPR biosensor generally employs a nanometer- thin layer (40−50 nm) of gold as a transducer. An incident light beam excites coherent oscillations of the metal conduction band electrons (i.e., surface plasmon polariton) that propagates along the interface metal-dielectric, generating an evanescent field that can extend up to a few hundreds of nanometers (10−300 nm) into the surrounding medium. When specific biorecognition elements (e.g., antibodies or DNA strands) are immobilized onto the gold surface, the selective capture and binding of the target molecule induce a change of the RI and in the light properties, which can be monitored and is directly proportional to the concentration of the analyte in the sample (Figure 2a). The RI limit of detection of SPR biosensors typically reaches 10−5−10−6 refractive index units (RIU), which commonly relates to detection limits in the low nM or even pM level in surface analyte detection. This analytical technique is consid- ered mature nowadays, and its potential has been vastly Figure 1. Biosensing strategies for virus infection diagnosis: (A) demonstrated by a myriad of applications in molecular biology detection of viral genomic material (i.e., DNA or RNA) by direct for the study of biomolecular interactions, in pharmaceutics for hybridization to a DNA probe; (B) detection of intact virus entities by the affinity and kinetics evaluation of drug candidates, and in antigen-specific recognition of antibodies; (C) detection of human environmental and biomedical diagnostics for the detection and antibodies against the virus antigens (serology assay). quantification of specific substances (i.e., small molecules, protein, nucleic acids, or pathogens) in different types of Both plasmonic sensors, based on metallic transducers (thin samples (e.g., food, water, human bodily fluids, etc.).51−53 In the gold films or nanostructures), and silicon photonics, fabricated past two decades, amid the rise of nanotechnology, plasmonic by conventional microelectronics technology, are in continuous biosensors have evolved to incorporate more sophisticated progress to move from working laboratory prototypes to nanostructures that can enhance the sensing performance and compact, integrated versions that can be deployed outside the improve miniaturization and integration capabilities. Nano- lab, near to the patient. These devices have already been patterned surfaces, such as arrays of nanoholes, nanodisks, or successfully applied for the diagnosis of pathogens, both bacteria nanorods, and more complex geometries like nanostars, and viruses, demonstrating their potential for this diagnostic nanodimers, or oligomer assemblies, have emerged as area.47,48 As discussed in the following sections, the technology interesting alternatives to conventional SPR for the develop- sensitivity and scalability, the selection of the receptors to target ment of ultrasensitive label-free biosensors as point-of-care the virus, and the appropriate biofunctionalization strategy on integrated devices (Figure 2b).39,43 Instead of propagating SPR, the sensor surface are crucial aspects to consider for these plasmonic nanostructures exhibit a localized resonance implementing successful diagnostic biosensors. ■ (i.e., localized surface plasmon resonance, LSPR) that is characterized by the higher confinement of the evanescent NANOPHOTONIC BIOSENSORS: WORKING field, with penetration depths around 10−50 nm, and by the PRINCIPLES AND TECHNOLOGIES spectral tunability of the resonance. Theoretically, the LSPR Most common nanophotonic transducers rely on nano- provides higher detection sensitivity, especially for relatively plasmonics or silicon photonics technologies. The underlying small targets, given that the biomolecular interaction occurs physics of these sensors have been widely studied and described within the entire depth of the evanescent field. The evidence of 2666 https://dx.doi.org/10.1021/acssensors.0c01180 ACS Sens. 2020, 5, 2663−2678 ACS Sensors pubs.acs.org/acssensors Review Figure 2. Schematic illustrations of the main label-free nanophotonic biosensor technologies: (A) Surface Plasmon Resonance (SPR) biosensor in Kretschmann configuration; (B) Localized Surface Plasmon Resonance (LSPR) biosensor based on gold nanodisks; (C) Mach−Zehnder interferometric (MZI) biosensor; and (D) ring resonator biosensor. this has been especially shown in Surface-Enhanced Raman plasmonic biosensors needs to incorporate and improve Spectroscopy (SERS) sensors, where the LSPR near-field is microfluidic systems that minimize or automatize sample exploited to largely intensify the Raman effect, providing processing, and user-friendly readouts that can be directly outstanding sensitivities that can even achieve single-molecule interpreted by nonspecialized users. detection.54 On the other hand, perhaps the most appealing Silicon Photonics Biosensors. Despite the predominance features of nanoplasmonic sensors rely on the capabilities for of nanoplasmonic biosensors as a reference in label-free optical spectral resonance design and more straightforward optical sensing, silicon photonics technologies have arisen as leading configuration possibilities. While propagating SPR generally platforms in terms of sensitivity and integration capabilities. requires complex light coupling schemes (e.g., prism-based Silicon photonics biosensors are based on integrated optical Kretschmann configuration), several LSPR sensing platforms waveguides fabricated on Si, Si3N4, SiON, or SiO2 materials. can work under normal light illumination, thereby simplifying Light travels along these microscopic waveguides under a TIR miniaturization and integration in small, portable, and user- regime, generating an evanescent electromagnetic field with friendly devices. This capability also facilitates the development penetration depths between 100 and 900 nm that is used as of two-dimensional sensing microarrays for multiplexed analysis. biosensing probe. The vast knowledge and robustness of silicon- Besides, the design and modeling of the nanostructured based technologies, including conventional microelectronics architecture enable the rational definition of the spectral peak fabrication and characterization procedures, together with the position at convenient wavelengths, so allowing for the use of low power consumption and the capability to incorporate all the affordable and widely available light sources, like light-emitting functions (chemical, optical, microfluidics, and electronics) in diodes (LEDs).43 one single platform, make these systems ideal for lab-on-a-chip Nanoplasmonic biosensor technologies, both SPR and LSPR implementation and use as point-of-care diagnostics.40,41 based, are nowadays available and commercialized worldwide. Most common Si photonics sensors are interferometers, ring In the case of SPR, large companies like GE Healthcare resonators, and photonic crystals, although other technologies (Biacore), Horiba, or Bruker, among others, deliver high-quality have also been developed, such as grating-coupled waveguides, benchtop systems that are routinely used in research laboratories silicon wires, slot waveguides, or optomechanical sensors, or the pharmaceutic industry. A few manufacturers also among others.41 Interferometric biosensors, like the Mach− commercialize LSPR-based instruments, such as Nicoya Life Zehnder (MZ), Young, or bimodal waveguide (BiMW) Sciences or LSPR AG. However, despite the advanced interferometers, stand out for their broad dynamic range and technological and analytical capabilities offered by these exceptional sensitivity, reaching detection limits in the range of instruments, their adoption in the clinical diagnostics field for 10−7−10−8 RIU.44,50 Figure 2c illustrates the working principle a given application is still very limited. This can be related to the of a MZ interferometric sensor. Briefly, an input waveguide is need for a robust, reproducible sensor surface biofunctionaliza- split into two arms (sensing and reference arms) that, after tion protocol, which usually must be optimized for each analyte, certain distance, are recombined into a single output. and the difficulties of analyzing complex samples like biological Biomolecular interactions occurring at the sensing arm within fluids. Furthermore, the achievement of truly POC nano- the evanescent field induce a phase difference with the light 2667 https://dx.doi.org/10.1021/acssensors.0c01180 ACS Sens. 2020, 5, 2663−2678 ACS Sensors pubs.acs.org/acssensors Review traveling along the reference arm. When recombined, the Table 1. Bioreceptors Applied for Virus Diagnosis generated interference provides the direct, label-free, and real- genomic detection time signal directly proportional to the analyte concentration. A variety of interferometric biosensors with different designs have receptor advantages limitations been developed and applied for clinical diagnosis purposes, DNA probes Stability and specificity Not sensitive enough in showing detection of small molecules,44 protein biomarkers or Easy to produce and attach some scenarios glycolipids,55 nucleic acids in the aM−fM range,56 or just a few to the surface pathogenic entities (e.g., 4−10 bacteria/mL).57 Tagged stem-loop probes Lower limits of detection Need for labels, chemical modifications Ring resonators, meanwhile, have attracted attention due to Additional steps their unique potential for integration into high-throughput Amplification + Lower limits of detection Additional steps arrays for multiplexed analysis,38,58 with highly efficient and DNA probes Cost of the enzyme, scalable fabrication. Ring resonator transducers consist of reagents circular waveguides that generate whispering-gallery modes intact virus detection (WGM) upon light coupling to the loop, enhancing the receptor advantages limitations evanescent field intensity (Figure 2d). Detection is performed Antibodies Robust and well established Animal requirement by interrogating the WGM spectral resonance position. The Wide range of ligands High production costs multiplexing capabilities of ring-resonator arrays have been High binding affinities and Activity decrease in long- demonstrated for the simultaneous detection of panels of selectivity term storage relevant diagnostic biomarkers, like cancer-related proteins or Low reusability microRNAs.38 Recombinant No animal requirement in the Need to know the sequence, Finally, photonic crystals also hold promising potential for antibody production or find binding regions fragments through phage display point-of-care biosensors due to their cost-effective fabrication Less costly to produce than Storage and reusability and high-throughput possibilities.59,60 Photonic crystals are the whole antibodies well-defined nanostructures with periodically repeated layers of (microorganism different refractive index. The lattice structure generates bioreactors) photonic bandgaps, highly dependent on the dielectric refractive Aptamers Possibility of engineering SELEX procedure is long Less costly than antibodies and complex index. These sensors have also been applied for biological assays achieving moderate sensitivities, compared to their silicon Stability and long-term storage photonics counterparts (in the nM−pM range), but they would Reusability greatly benefit from an easy and low-cost mass production of Glycans Less cost in production Limited to a few viruses disposable photonic sensor chips. Increase selectivity in Need of prior studies of Silicon photonic technologies have also started recently to hit combination with other glycan affinities to viruses the biosensors market with some successful systems like the ring receptors resonator-based platform supplied from Genalyte. However, serological assay they suffer from the same shortcomings as nanoplasmonic receptor advantages limitations technologies in terms of surface biofunctionalization and Viral lysates Easy to produce Not homogeneous receptor integration in automated and user-friendly lab-on-a-chip devices layers for their implementation in the clinical field. Extended No need to know the viral Cross-reactivity due to discussion in this regard is provided in the following sections antigens or genome incorporation of host sequence proteins in virions of this review. Recombinant Homogeneous receptor Need to identify the gene ■ viral antigens layer sequence BIORECEPTORS AND SENSOR SURFACE Relatively easy to produce Cross-reactivity with strains of the same family BIOFUNCTIONALIZATION Antigenic Less cross-reactivity with Need to know sequence, The performance and, especially, the sensitivity and selectivity of domains of viruses of the same family structure, and identify the viral proteins domain surface-based nanophotonic transducers will ultimately depend on the most appropriate biorecognition element that binds specifically the target analyte, and on the conditions for the For the case of genomic hybridization assays, oligonucleotide coating of this receptor. As for any application to be developed, capture probes are designed and synthesized with the bioreceptors are selected according the approach for detection complementary sequence to the specific viral target region. of the target. In the case of viral infection diagnosis, considering Primarily, the specificity will depend on the sequence selection the three main strategies described before, these would be DNA of the probe and, second, on the hybridization conditions, capture probes for nucleic acid detection, specific receptors like mainly buffer composition and temperature. Aspects to be antibodies for antigen-directed virus capture, or viral antigens considered when designing the probe will be the pair length and for serological tests. However, many variants and innovative the nucleotide sequence, because they will affect the binding bioreceptor elements have been developed to enhance the energy and stability of the complex. In addition, especially in sensing performances (see Table 1). It is important to note RNA detection, secondary structures around the targeted zone though that the access to high-quality and well-characterized might hinder the binding to the probe, and then, it is essential to biological reagents, especially antigens and antibodies, can be a select regions where these folds are not formed.61 For some significant problem, in particular at the start of an outbreak from cases, where the biosensor sensitivity is not enough, the use of a new pathogen, like the SARS-CoV-2. This inherently delays tagged hairpin probes or the hybridization of additional labeled the development of reliable sensor technologies as well as other sequences has been prevalent as amplification steps. For diagnostic approaches, such as the RADTs, as discussed before. instance, stem-loop oligos tagged with signal amplification 2668 https://dx.doi.org/10.1021/acssensors.0c01180 ACS Sens. 2020, 5, 2663−2678 ACS Sensors pubs.acs.org/acssensors Review compounds (e.g., SERS reporters) have been used to improve surface (e.g., gold or silicon derivatives) can be done in different the detection limit in the analysis of different human viruses.62 ways: by direct chemical or physical adsorption, by covalent For antigen-directed virus detection, most common bio- linking to a chemical matrix, and through affinity binding to receptors are monoclonal antibodies, which can be produced to certain molecules. Physical adsorption through electrostatic and target specific viral antigens and have been extensively used in hydrophobic interactions is not recommended, as it can lead to biosensor development. In the past decades, smaller recombi- biomolecule denaturation, and very low stability and reprodu- nant antibody fragments and engineered variants have become cibility. Certain biomolecules, however, can be directly advantageous alternatives.63 These peptides can be selected by chemisorbed to the surface, for example, via thiol (-SH) binding phage display, which allows screening of clone libraries of to gold.84 This is widely employed for oligonucleotide probe recombinant phages expressing on their surface the binding functionalization (DNA probes or aptamers). It can also be used regions of antibodies and proteins. The selected ones can be with proteins by the cysteine residues, although in some cases, it identified and produced synthetically or expressed in bacteria, can lead to the wrong orientation of the molecules.85 with fewer requirements than the production of antibodies in Covalent immobilization involves the chemical modification hybridomas or mammal cells. As an alternative, aptamers, which of the transducer surface to generate a functional chemical are short DNA/RNA sequences that also bind with high affinity matrix (e.g., self-assembled monolayers, SAMs), where bio- and specificity to target molecules, have also been proposed for molecules can be covalently bound. Compared to the previous the detection of viral antigens, showing sensitivities comparable approaches, it offers some advantages: a more uniform and to monoclonal antibodies.64−66 Main advantages are related to controlled surface coverage, possibility for long-term storage, the simpler production and possibility to introduce any desired and sensor reusability. The SAMs are created by distinct functionality for surface immobilization. However, the selection compounds depending on the transducer material, e.g., procedure is not yet well established to be massively alkanethiol molecules for gold (which involves very well commercialized and employed with sufficient robustness. At established and reproducible procedures) and alkosysilanes for present, many aptamers have been isolated for the recognition of Si-based sensors.85,86 The silanization procedure, although also human viruses,67,68 and particularly for respiratory viruses; most widely used, is relatively complex, and many factors influence its of them have been designed to detect distinct strains of reproducibility, such as the silane concentration, solvents influenza.69−71 Finally, cell receptors have also been proposed employed, reaction times, or temperature. The molecules that for direct virus detection, as the case of glycans from the host comprise either type of SAMs can be engineered to have cells. Surface receptor glycans have been, in fact, widely used in different lengths and terminal groups that allow the grafting of conjunction with biosensors for the detection of different strains the probes. The most common SAMs terminations are amine of influenza.72−74 Moreover, the combination of glycoprotein (NH2), thiol (SH), carboxyl (COOH), and epoxy (COC) receptors and antibodies has improved the specificity of the functionalities that can be cross-linked with native or engineered sensor to certain viral strains.75 functional groups in the biomolecules. Given the broad range of For serological assays, the bioreceptors essentially consist of linkers used depending on terminal groups, we invite the reader viral antigens, the same that cause the host immunogenic to check other reviews and chapters for a more in-depth reaction.76,77 The first serological tests employed whole virus classification of compounds, protocols, advantages, and draw- lysates for the recognition of the host antibodies, resulting in test backs.85,87−89 Finally, affinity-based immobilization strategies variability and false positives due to the presence of host proteins are also commonly employed for bioreceptor immobilization, in the virions.78 Since then, advances in cloning, genetic with streptavidin−biotin coupling the more employed.90 This engineering, and the establishment of robust expression systems strategy involves streptavidin coverage of the surface by make possible the use of recombinant viral antigens instead. adsorption or covalent linkage and the addition of the Once the genome of different viral strains is available, biotinylated receptor. This approach requires the labeling of recombinant proteins are produced at large scale in bioreactors, the receptor with biotin groups but provides a highly stable and facilitating the production of homogeneous reactive layers. By specific bond. Another affinity-based immobilization strategy using cloned proteins, large amounts of purified antigens can be widely used, especially for antibodies, involves the use of Protein produced and isolated with less cost in addition to reducing the A or G, which binds with high affinity to the Fc region of risk of infection.79 In some cases, the whole viral protein is not antibodies. This methodology provides an efficient strategy to necessary since only a part of the protein causes most of the orientate the antibodies to the outer medium, leaving more immunogenic reaction in the individuals. Then, the production exposed the regions responsible for the specific binding.91 of this small region (which can be a short peptide) may be Lastly, there are two other critical aspects to consider: (i) enough, simplifying the process. Compared to the whole ensuring adequate bioreceptor density, minimizing possible recombinant protein, these shorter peptides are designed to steric hindrance effects, and (ii) avoiding nonspecific include nonconservative regions, so they have fewer cross- interactions on the sensor surface. In the first case, it is necessary reactions with sera raised against similar viral infections,80,81 to study the effect of combining mixed SAMs (with reactive and thus increasing the specificity. These short peptides are nonreactive) groups, which help space out the distribution of habitually conjugated to larger proteins, known as carrier receptors (particularly important if they are large molecules, like proteins, to increase stability and facilitate surface immobiliza- are bacteria or viruses) or also spacers that move away the tion.82,83 receptor from the sensor surface (i.e., increasing their Besides the bioreceptor selection and its quality, a critical accessibility). On the other hand, nonspecific interactions, factor for the biosensor performance is the appropriate surface often referred to as “fouling”, are a critical issue when analyzing functionalization and its immobilization, taking into account clinical samples such as saliva, sputum, urine, serum, or blood, as orientation, stability, reproducibility, and prevention of non- they can induce misleading signals (i.e., false positives resultant specific adsorption of matrix components of the biological of binding to the sensor surface and change in the refractive samples. The bioreceptor immobilization to the transducer index) and might also hinder the recognition of low- 2669 https://dx.doi.org/10.1021/acssensors.0c01180 ACS Sens. 2020, 5, 2663−2678 ACS Sensors pubs.acs.org/acssensors Review Table 2. Nanophotonic Biosensors Applied for Respiratory Virus Diagnosis genomic detection biosensor technique virus receptor target sample sensitivity ref LSPR SARS-CoV-2 DNA probe RNA Buffer 0.2 pM 98 Microring Influenza A and B, hCoV-OC43/229E, RSV A and B DNA probe RNA Nasopharyngeal 25 copies/ 99 Resonators samples reaction SPR Influenza A and B, H1N1, RSV, parainfluenza, adenovirus, DNA probe RNA Buffer 1−5 nM 100 and SARS-CoV SERS High-pathogenic avian influenza (HPAI) Raman-tag hairpin DNA RNA Buffer 2−3 aM 62 probe intact virus detection biosensor technique virus receptor target sample sensitivity ref SPR Avian influenza H6N1 Antibodies H6N1 virion Buffer 5 × 106 EID50/mL 103 SPR Avian influenza H7N9 Antibodies HA protein Buffer 200 copies/mL 104 SPR Enterovirus 71 Antibodies VP1 protein Culture media 67 virus particles/mL 105 SPR Avian influenza H5N1 Aptamer HA protein Poultry swab samples 0.128 HAU/mL 70 SPR Avian influenza H5Nx Aptamer HA protein Buffer 200 EID50/mL 69 Waveguide interferometer High-pathogenic avian influenza (HPAI) Antibodies HA protein Buffer 0.0005 HAU/mL 106 Silica Inverse Opal Avian influenza H1N1 Antibodies HA protein Buffer 103 PFU/mL 107 Photonic Crystal Avian influenza H1N1 Antibodies HA protein Saliva samples 1 ng/mL 108 Mach−Zehnder Avian influenza H1N1 Antibodies HA protein Buffer 1010 virus particles 109 Figure 3. (A) Example of nanophotonic biosensor applied for COVID-19 diagnosis via genomic detection of SARS-CoV-2. Reprinted (adapted) with permission from ref 98. Copyright (2020) American Chemical Society. (B) Example of nanophotonic biosensor applied for direct detection of intact viruses (influenza). Reprinted (adapted) with permission from ref 104. Copyright (2018) American Chemical Society. (C) Example of smartphone- integrated optical biosensor. Reprinted (adapted) with permission from ref 130. Copyright (2017) Elsevier. (D) Example of microfluidics-integrated optical biosensor. Reprinted (adapted) with permission from ref 134. Copyright (2014). American Chemical Society. Please refer to original articles for reprint. concentration analytes.92 There is extended research devoted to compounds while preserving the affinity and sensitivity of the study the factors influencing these interactions and to develop recognition elements. These coatings have been able to avoid strategies to counteract them. The antifouling strategies involve the fouling attachment by the creation of hydration layers and the use of compounds that prevent the adsorption of other electrostatic and steric repulsions.93 Materials that have shown 2670 https://dx.doi.org/10.1021/acssensors.0c01180 ACS Sens. 2020, 5, 2663−2678 ACS Sensors pubs.acs.org/acssensors Review excellent antifouling properties include polyethylene glycol analysis. For example, Pang et al. developed a SERS sensor (PEG), zwitterionic polymers, polysaccharides such as dextran, combined with a molecular sentinel for the detection of the and proteins like bovine serum albumin (BSA).94 avian influenza virus. They reached detection limits around 2−3 ■ NANOPHOTONIC BIOSENSORS FOR VIRUS DETECTION aM, allowing a PCR-free analysis method.62 As major assets compared to standard RT-PCR technique for viral genomic analysis, these nanophotonic biosensors account The potential and versatility of label-free nanophotonic with the direct and label-free capabilities that reduce total biosensors for medical diagnostics have been widely demon- turnaround analysis time from several hours (4 to 6) to less than strated, including virus detection and identification, especially 30 min. Potential applications of these technologies for the for the human immunodeficiency virus (HIV) and the hepatitis COVID-19 pandemic and possible future outbreaks could B virus.47,94−97 Hereby, we provide a brief review of the latest signify the realization of truly reliable massive testing and developments in nanophotonic biosensors for respiratory virus screening. By targeting appropriate gene sequences and infection diagnosis based on the three main assay strategies providing ultrasensitive analysis, the genomic biosensing assays described above. can provide highly accurate and specific detection of the virus, Nanophotonic Biosensors for Viral Genomic Analysis. even when viral load is low. Furthermore, by establishing Detection of specific genomic sequences with label-free multiplexed genomic analyses, it could be possible to identify photonic biosensors is usually carried out through rapid different types of viruses or strains in one single assay. hybridization assays. If the biosensor is sensitive enough, direct Nanophotonic Biosensors for Antigen-Directed Virus detection from the sample might be possible, without Detection. The first works for intact virus detection with label- preamplification steps (PCR-free), although it still requires free optical biosensors were reported by Schofield and Dimmock extraction and fragmentation procedures to be performed before around 25 years ago.101 They employed an SPR biosensor to the biosensing assay. determine the affinities of different antibodies and Fab Due to the intricate bioassay design and procedure, only a few fragments for influenza A viruses by monitoring the capture of biosensors have been developed for the genomic assay of whole virions. Since then, many researchers have been respiratory viruses (see Table 2). At the time of writing this developing new biosensor assays for rapid virus detection and review, at least one nanoplasmonic biosensor has been reported quantification with plasmonic and photonic technologies (see for the detection of the SARS-CoV-2 virus. Qiu et al. have Table 2). The vast majority of the work in respiratory virus developed a dual-mode nanoplasmonic system, consisting of diagnosis targets influenza viruses and the different subtypes.102 two-dimensional gold nanoislands, that combines plasmonic In recent publications, for example, Zhao et al. reported an photothermal effect and LSPR sensing transduction (Figure optical SPR fiber sensor for the detection of avian influenza 3a).98 The sensor is functionalized with a DNA probe of a virus.103 With a compact and low-cost system, they were able to complementary sequence to the RdRp RNA gene of the detect around 5 × 105 EID50 in 100 μL sample within 10 min. COVID-19 virus. The specific nucleic acid hybridization, Chang et al. also employed an SPR biosensor for avian influenza enhanced by the thermoplasmonic heat generated at the detection with a newly generated antibody (Figure 3b).104 They nanoislands, is detected as a LSPR phase shift that can be reached a detection limit of around 200 copies/mL, which monitored in real-time. They achieved a limit of detection resulted in 20-fold improved sensitivity compared to analogous around 0.2 pM for the target RNA sequence, which could be ELISA tests, and better than conventional RT-PCR and rapid translated into approximately 2 × 104 copies of the SARS-CoV-2 diagnostic tests. Likewise, Prabowo et al. proposed a portable virus, showing good potential for COVID-19 diagnostics. SPR biosensor for the rapid quantification of enterovirus Previous studies have also addressed the genomic analysis of antibodies targeting the VP1 membrane protein.105 The SPR viral infections. Koo et al., for example, developed a label-free system showed a detection limit of 67 virus particles per biosensor using silicon microring resonators for the diagnosis of milliliter. Besides antibodies, aptamers have also been applied respiratory viral infections. They performed an isothermal RNA for avian influenza virus detection. Bai et al. developed an SPR amplification coupled to the hybridization biosensing assay for aptasensor with detection sensitivities around 105.70 A more the detection, all within 20 min.99 The biosensor was applied for exhaustive study was reported by Nguyen et al., including the the analysis of nasopharyngeal samples from patients infected selection of aptamer pairs to develop a sandwich-type SPR assay with influenza A and B, human coronavirus OC43/229E, and for avian influenza virus detection.69 In this assay, they reached RSV A and B, respectively. The limit of detection was found sensitivities around 200 EID50/mL. close to 2.5 × 101 copies/reaction and clinical sensitivities Apart from plasmonics, other nanophotonic sensors have also between 85% and 100%, although specificity was low for some been applied for direct virus detection. Xu et al. developed a viruses (40−60% for hCoV) probably due to the lack of enough waveguide-based interferometric biosensor for the direct samples. This biosensor exhibited a reliability comparable to detection of influenza viruses.106 They tested different that of conventional RT-PCR, reducing the analysis time to a monoclonal and polyclonal antibodies targeting the HA protein few minutes and avoiding additional fluorescence or electro- of the membrane of influenza viruses. Their biosensor phoresis-based detection methods. With a similar aim, Shi et al. immunoassay reached outstanding sensitivities, with a detection reported an SPR biosensor for multiplexed RNA detection of limit of 0.0005 HAU. Lee et al. fabricated a label-free optical several respiratory viruses: influenza A and B, H1N1, RSV, sensor based on three-dimensional inverse opal silica nano- parainfluenza virus, adenovirus, and SARS coronavirus simulta- structures.107 Their application for direct avian influenza virus neously.100 However, a previous PCR amplification of the target detection by antibody capturing rendered sensitivities in the genetic material plus a secondary streptavidin-amplification step range of 103−105 PFUs (plaque forming units). Endo et al. after hybridization were required. Overall, sensitivities achieved developed a flexible two-dimensional photonic crystal biosensor were in the range of 1−5 nM. Other nanoplasmonic approaches employing antibodies targeting avian influenza virus HA like SERS have also been applied for respiratory virus genomic protein.108 They reached a limit of detection of 1 ng/mL for 2671 https://dx.doi.org/10.1021/acssensors.0c01180 ACS Sens. 2020, 5, 2663−2678 ACS Sensors pubs.acs.org/acssensors Review direct measurement in human saliva samples. Finally, Sakamoto the other hand, Zhang et al. developed a novel arrayed imaging et al. employed a Mach−Zehnder interferometer for avian reflectometry platform for the high-throughput study of the influenza diagnostics.109 They only tested the technology for the serologic antigenicity of influenza viruses.121 detection of 1010 virus particles that showed a clear signal It is important to note as well that nanophotonic biosensors response in comparison with the negative control. have shown exceptional utility in different virology and The availability of sensitive and reliable intact virus detection immunology studies. Nilsson et al., for instance, published an tests for SARS-CoV-2 could transform the outbreak status, SPR biosensor for quantification of influenza virus HA protein management, and progression. The implementation of bio- based on an inhibition assay.122 They immobilized the HA sensors for rapid screening of the population and immediate proteins for different virus subtypes on the sensor surface and detection of COVID-19 would allow more efficient patient quantified the levels of free specific antibodies in the sample, isolation and spread control. Nanophotonic biosensors have after incubation with the infected sample. The limit of detection demonstrated superior sensitivity and robustness compared to was around 0.5 μg/mL, but they were able to identify the LFA tests, plus they offer quantitative results in approximately different avian influenza virus subtypes and strains in serum the same turnaround assay times. Ongoing research is focusing samples. Negri et al. used label-free SERS sensors for the on developing and applying these valuable technologies for detection of viral nucleoprotein binding to an anti-influenza direct and rapid diagnostics, and especially for the point-of-care aptamer.71 Although they did not reach a high sensitivity (1 μg/ testing of coronavirus infection. A good example is one of the mL), they could demonstrate specific detection of the influenza first and largest research projects funded by the European nucleoprotein challenged to other respiratory viruses and Commission to tackle the COVID-19 pandemics (i.e., CoNVat biological samples. On the other hand, Vidic et al. used an project), which will employ a silicon interferometric sensor for SPR biosensor to identify and characterize a novel complex intact virus detection in nasopharyngeal and saliva samples.110 between the nucleoprotein and the RNA polymerase of the Besides, a few reports have been uploaded in non-peer-reviewed influenza A virus.123 This molecular complex has a role in the repositories showing proof-of-concept studies. For example, final steps of transcription and replication of the virus, so it could Ahmadivand et al. have developed a plasmonic metasurface for be a target for therapeutics. Xiong et al. applied biolayer sensing the S protein at the femtomolar level.111 On the other interferometry technology to measure the binding affinity of hand, Zhang et al. employed silver nanorod SERS sensors for human and influenza viruses to their cell receptors.124 ultrafast detection of viral antigens (RBD). The technology has In the coronavirus area, some studies have also been been applied for water monitoring but could also be adapted for performed with optical biosensors. After the last coronavirus diagnostics.112 outbreaks (SARS and MERS), researchers have continued Nanophotonic Biosensors for Serology and Immunol- studying and characterizing the virus structures and searching ogy Assays. Although not considered a primary diagnosis for potential drugs and therapeutic agents. Huang et al., for strategy, serology and immunology assays have a strong example, studied the evolution of the receptor-binding domain relevance in an infection disease prevalence study and the (RBD) of the Spike protein of β-coronavirus and its interaction design and assessment of vaccines and other therapeutics. with cell receptors, employing an SPR platform to characterize Optical label-free biosensors offer a unique technique to the affinity and kinetics of the interaction.125 Likewise, Park et al. facilitate, accelerate, and improve the routine pharmaceutical evaluated different polyphenols as coronavirus protease and biomedical analysis. inhibitors with an SPR biosensor.126 They could select one of Serology biosensing assays are relatively easy to implement, as these molecules as a promising candidate for developing they essentially consist of direct quantification of antibodies anticoronaviral agents. through binding to their corresponding antigen immobilized on The use of plasmonic and photonic biosensors in serology and the sensor surface. Scarcely any articles have been recently immunology studies has undoubtedly benefited the pharma- published for the development and validation of such serological ceutical industry and biomedical research. It could soon replace biosensors for respiratory viruses.113,114 However, the emer- long and tedious ELISA, offering a faster, simpler, and even gence of SARS-CoV-2 and the urgent need for massive testing cheaper technique. For the COVID-19 case, serological analyses has promoted the application of nanophotonic sensors for have become crucial not only as a complementary diagnosis but COVID-19 serology with diagnosis and monitoring purposes. also for pandemics management and epidemiology studies. It is Very recently, Shaw et al. have published a clinical evaluation now essential to perform massive tests for the whole population study of a nanoplasmonic technology for quantitative detection to understand the incidence and mortality of the disease, and the of SARS-CoV-2 antibodies in different disease stages.115 Other effects on human health and immunity. For the development examples can be found in repositories. Abdelhadi et al. have also and evaluation of new vaccines or other therapies (e.g., developed a SPR bioassay for SARS-CoV-2 antibody testing,116 hyperimmune serum treatments), it is crucial to accurately and Kulp et al. demonstrate the utility of SPR biosensors for study the generation and kinetics of sera antibodies, and rapid antibody neutralization assays, studying recognition of the optimally in a quantitative and highly specific way. For that, ACE2 receptor.117 label-free nanophotonic biosensors are promising and realistic solutions. ■ On the other hand, many research works in vaccine development and evaluation employ plasmonic and photonic biosensors for characterization and evaluation procedures. For FUTURE PERSPECTIVES example, Fuentes et al. characterized the affinity and kinetics of Nanophotonic biosensors have demonstrated their detection antibodies against RSV generated during vaccine development, a performance and capabilities for clinical diagnostics, including key step in preclinical evaluation.118 Marsh et al. employed a discrete microorganisms such as micrometer-sized bacteria and SPR system to assess the susceptibility of RSV to antifusion small viruses on the nanometer scale. The detection of protein antibodies,119 and Gauger et al. also used it for pathogens that tackle the drawbacks of current methodologies evaluation of live attenuated influenza A virus vaccine.120 On is possible with the high levels of sensitivity this technology can 2672 https://dx.doi.org/10.1021/acssensors.0c01180 ACS Sens. 2020, 5, 2663−2678 ACS Sensors pubs.acs.org/acssensors Review reach. They can be used for the detection of antibodies in serum, detection of tuberculosis in urine samples.55 Using automated the detection of DNA and RNA fragments with excellent pneumatic-valve based microfluidics144−146 or digital micro- performance, avoiding amplification, and even for the detection fluidics147,148 instead of conventional microchannel-based of whole viruses, which shows the versatility to implement such fluidic chips represents an emerging trend to be used with tools in the diagnosis of respiratory viruses such as SARS-CoV-2. silicon photonics given its potential to be integrated in However, there is still much effort to be done, especially on miniaturized design and its versatility to transport and mix two fronts. First, to systematically perform rigorous clinical very small volumes with high precision, thus being very attractive validation of each individually optimized assay to fully assess the for multiplexed automated analysis. However, scarce examples reliability and robustness of the detection strategy. This is not are found in the literature149 and not tested further with always provided as it involves the evaluation of a high number of biosensors. Despite the excellent biosensing performance of real samples to determine specificity and sensitivity statistics nanophotonic biosensors, there is still a long road ahead in parameters. Second and most importantly, we need a strong several technological aspects to achieve fully operative silicon push to move the technology from laboratory prototypes, and plasmonic devices for clinical diagnosis. The current commonly installed in a controlled and robust environment, to emergency situation, which has revealed the shortage of really decentralized settings. This step involves the design of all- effective diagnostic techniques, may represent an inflection integrated, autonomous, compact devices, incorporating optical point to push further the research and investment in optical elements that facilitate both the reduction in size and in cost, biosensors toward such levels of integration necessary to while retaining the performance achieved in laboratory versions, successfully hit the market. ■ to further pursue viable commercialization. In this sense, highly advanced smartphone technology offers excellent and promising SUMMARY AND CONCLUSIONS platforms to design point-of-care versions (Figure 3c).127−130 The outbreak of COVID-19 has dramatically exposed the urgent For example, an optical interface based on complementary need to upgrade the clinical diagnostics to implement new metal-oxide semiconductor (CMOS) technology in the phone technologies for POC testing with sufficient accuracy and cameras for readout, in combination with customized software reliability. Nanophotonic biosensors have demonstrated in implemented via smartphone applications, can be used for academic settings the capabilities for such endeavor, offering a different optical detection approaches, from labeled approaches unique potential to combine rapid virus detection and (like colorimetry and fluorescence) to label-free detection, identification, genomic analysis, and even serological assays in through imaging readout or spectrum monitoring.131 Additional one integrated platform. Key factors for the successful components such as external light sources, like affordable LEDs implementation of label-free nanophotonic biosensors in virus or broadband lights, collimators, or polarizers, can be included in infection diagnosis are essentially the selection of appropriate an adapted miniaturized design, involving also compatible and and high-quality bioreceptors, which confer enough sensitivity suitable customized polymer shell or housing to merge with the and selectivity, together with a robust surface biofunctionaliza- cell phone. More examples are appearing trying to combine tion. The rational design and optimization of label-free smartphone technology with plasmonic biosensors,132,133 nanophotonic biosensors can provide direct detection of intact although the performance is still undermined compared with virus entities or specific genomic identification directly from a robust laboratory versions and they have been tested with model human sample with outstanding sensitivities (few copies per biosensing systems or with limited analytical characterization. milliliter). Further, the simplicity and efficiency for routine and Microfluidics also play a crucial role to provide disposable, fast serological analysis could significantly contribute to stable over time, easy-to-manipulate cartridges incorporating the epidemiology studies or vaccine development. Henceforth, sensor chip with the specific biofunctionality for each detection research in nanophotonic technologies for diagnostic biosensors assay.135−137 In the case of airborne respiratory viruses, like should move a step forward to prove their capabilities in the SARS-CoV-2, it will be essential to integrate such a cartridge clinical field and provide fully operative point-of-care devices for with additional sections that include sample preprocessing steps rapid, accurate, and massive infection diagnosis. before the final detection.138 Some of these steps might include extraction of the viral genome from the clinical samples,139 separation of plasma, or serum from blood134,140−142 whose direct measurement is exceptionally challenging with optical ■ AUTHOR INFORMATION Corresponding Author biosensors, or even more appealing, oral samples pre-treatment Maria Soler − Nanobiosensors and Bioanalytical Applications (nasopharyngeal swabs, saliva, sputum, etc.).143 In the case of (NanoB2A), Catalan Institute of Nanoscience and oral samples, viral load is expected to be high, and minimum Nanotechnology (ICN2), CSIC, BIST and CIBER-BBN, 08193 manipulation would facilitate the prevention of healthcare staff Bellaterra, Barcelona, Spain; orcid.org/0000-0001-7232- infection as well as further expansion of the device use outside of 2277; Email: maria.soler@icn2.cat biosafety settings (e.g., primary care centers). There are plenty of examples of microfluidics that perform filtration, extraction, Authors and centrifugation for preconcentration, and more complex Maria Carmen Estevez − Nanobiosensors and Bioanalytical processes such as nucleic acid amplification. However, they are Applications (NanoB2A), Catalan Institute of Nanoscience and commonly coupled colorimetric or electrochemical detections Nanotechnology (ICN2), CSIC, BIST and CIBER-BBN, 08193 and it is rarely reported for label-free optical biosensors. Some Bellaterra, Barcelona, Spain; orcid.org/0000-0003-3694- interesting examples have already been proposed merging 7186 microfluidics integration and nanophotonic biosensors (Figure Maria Cardenosa-Rubio − Nanobiosensors and Bioanalytical 3d). For example, an individual polymeric cartridge with an Applications (NanoB2A), Catalan Institute of Nanoscience and interferometric photonic chip that can be directly inserted in the Nanotechnology (ICN2), CSIC, BIST and CIBER-BBN, 08193 POC device, which has been validated for the pump-free direct Bellaterra, Barcelona, Spain 2673 https://dx.doi.org/10.1021/acssensors.0c01180 ACS Sens. 2020, 5, 2663−2678 ACS Sensors pubs.acs.org/acssensors Review Alejandro Astua − Nanobiosensors and Bioanalytical immunoglobulin is a blood-circulating protein produced by Applications (NanoB2A), Catalan Institute of Nanoscience and the immune system of an organism to recognize and neutralize Nanotechnology (ICN2), CSIC, BIST and CIBER-BBN, 08193 specific antigenic molecules or pathogens; Serology: analysis Bellaterra, Barcelona, Spain and study of the blood serum composition for diagnostic Laura M. Lechuga − Nanobiosensors and Bioanalytical purposes, especially with regard to the identification of immune Applications (NanoB2A), Catalan Institute of Nanoscience and system response (i.e., antibody production) to pathogenic Nanotechnology (ICN2), CSIC, BIST and CIBER-BBN, 08193 agents or other antigenic substances ■ Bellaterra, Barcelona, Spain; orcid.org/0000-0001-5187- 5358 REFERENCES Complete contact information is available at: (1) Troeger, C.; Blacker, B.; Khalil, I. A.; Rao, P. C.; Cao, J.; Zimsen, S. https://pubs.acs.org/10.1021/acssensors.0c01180 R. M.; Albertson, S. B.; Deshpande, A.; Farag, T.; Abebe, Z.; et al. Estimates of the Global, Regional, and National Morbidity, Mortality, Author Contributions and Aetiologies of Lower Respiratory Infections in 195 Countries, ‡ M.S. and M.C.E. contributed equally. The manuscript was 1990−2016: A Systematic Analysis for the Global Burden of Disease written through contributions of all authors. All authors have Study 2016. 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