key: cord-1048807-9wkf1hy8 authors: Ribeiro, Brayan Viana; Cordeiro, Taís Aparecida Reis; Oliveira e Freitas, Guilherme Ramos; Ferreira, Lucas Franco; Franco, Diego Leoni title: BIOSENSORS FOR THE DETECTION OF RESPIRATORY VIRUSES: A REVIEW date: 2020-08-16 journal: Talanta Open DOI: 10.1016/j.talo.2020.100007 sha: cbd4cddc07dcb5cdeafe9d227f382135f11e1afe doc_id: 1048807 cord_uid: 9wkf1hy8 Abstract The recent events of outbreaks related to different respiratory viruses in the past few years, exponentiated by the pandemic caused by the coronavirus disease 2019 (COVID-19), reported worldwide caused by SARS-CoV-2, raised a concern and increased the search for more information on viruses-based diseases. The detection of the virus with high specificity and sensitivity plays an important role for an accurate diagnosis. Despite the many efforts to identify the SARS-CoV-2, the diagnosis still relays on expensive and time-consuming analysis. A fast and reliable alternative is the use of low-cost biosensor for in loco detection. This review gathers important contributions in the biosensor area regarding the most current respiratory viruses, presents the advances in the assembly of the devices and figures of merit. All information is useful for further biosensor development for the detection of respiratory viruses, such as for the new coronavirus. At the end of 2019, the whole world was affected by a new pandemic, for early detection, like biosensors, an easy-to-use device that can rapidly detect the disease even in asymptomatic conditions with high reliability and low cost. One effective way, reported at media briefing from WHO Director-General to slow down the virus dissemination during the 2019 Covid-19 pandemic, was the social isolation, recommendation adopted worldwide [3] . The most effective way to this date, to detect the virus is the use of real-time quantitative polymerase chain reaction (RT-qPCR) [4] , while antibody-based techniques (IgG/IgM) are being introduced as supplemental tools [5] . So far, expensive, time-consuming detection systems with the need for skilled labor personal are being used and evaluated. Moreover, during the social isolation, the individual need to break out from the confinement to perform the medical test, which increases the risk to enter into contact with the virus in the process. Therefore, the development of biosensors is important because the device can be operated in loco and it is ready-to-use by any personal. This review focus on the most current contributions of biosensors designed for respiratory virus detection. The world scenario with the COVID-19 pandemic and the concerns regarding the latest and worrying outbreaks makes this survey of great importance for researchers planning to develop strategies for fast diagnosis. Some papers developing biosensors for SARS-CoV-2 determination have already been published, which proves that the biological material is already at hand to researches for further studies regarding this subject. Biosensors are analytical devices that convert biological reactions into measurable signals. The biological material such as enzymes, tissues, microorganisms, antibodies, cell receptors, or a biomimetic component, is immobilized over a transducer, and interacts with the analyte in the solution, producing a biochemical response (Fig.1 ). The transducer, in turn, converts this biochemical response into a quantifiable signal measured by the digital detector module [6, 7] . The main types of transducing systems are the electrochemical, optical, and piezoelectric. Electrochemical biosensors monitor alterations in charge distribution over the transducer surface, based on potentiometric [8, 9] , amperometric [10] [11] [12] or impedimetric [13] [14] [15] transduction principles. Optical biosensors are versatile tools for analytical purposes because it provides multiplexed detection within a single device. These devices focus on the measurement of optical properties and characteristics of the transducer surface when occurring the interaction of the analyte with the recognition element. [16] [17] [18] [19] . Piezoelectric biosensors employ transducers that resonate when an external alternating electrical field is applied. They are based on the measurement of changes in the resonance frequency caused by the mass of the crystal and the immobilized biological material. According to the corresponding variation of electrical signal upon contact with the analyte, the difference in mass can be assessed. Among the many applications, these biosensors have been used in a wide in medicine to detect targets in biological systems [20] [21] [22] . The fast detection of biological pathogens plays a crucial role in the prevention of disease spread, infections, and pathologies [23] . Biosensors have found immense applications in medical diagnostics, and it offers more specific, sensitive, fast, and reproducible results as compared to the conventional techniques like biochemical assays and immunoassays [24] . Moreover, biosensors are been increasingly applied in clinical analysis due to their portability and point of care testing, which can analyze real biological samples in routine clinical use [25] . The incorporation of nanotechnology in the design of biosensors has improved the detection of biological specimens, as the preparation of biointerfaces of self-assembled monolayers (SAMs), improving biocompatibility and resistance against nonspecific adsorption [26] [27] [28] [29] . Point-of-care testing (POCT) is one of the most important applications of biosensors for infection diagnosis. POCT measurement is the practice of performing a diagnostic test near the patient to provide rapid results, providing appropriate, convenient care to patients, and more effective treatment of rapidly progressing infections. Moreover, these devices can be used without expensive instrumentation [30, 31] . Accurate and early diagnoses play a crucial role in identifying the actual cause and nature of any disease. Currently, the focus shifted toward the early detection of COVID-19 disease. Saliva has a pivotal role in non-invasive salivary diagnostics that provide a convenient and cost-effective POCT platform for fast detection and may be an attempt to improve the chances of survival of patients from COVID-19 disease [32, 33] . Biosensors can be further classified according to the analytes or reactions that they monitor as immunosensors (antibody-antigen interaction), enzymatic biosensors (enzyme-target analyte interaction) DNA biosensor (hybridization) and whole-cells biosensor. Immunosensors are biological sensors based on the specific interaction between antibodies and antigens. The lymphocyte B produces the antibody upon the host contact with an antigen and performs clonal expansion and differentiation. After, the antigens are eliminated, followed by the apoptosis of effector lymphocytes and remaining of memory B cells [34] . It has been developed for continuous monitoring of analytes through point-of-care devices, which provide low cost, full automation, portability, fast response, high sensitivity, accuracy, and precision [35] . The application of immunosensors in clinical diagnosis and monitoring of diseases has been emphasized in recent works and it is mainly reported for the detection of biomarkers [36, 37] hormones [38] [39] [40] , pathogenic bacteria [41] [42] [43] [44] [45] , viruses [46] [47] [48] [49] and toxins [50] [51] [52] [53] . Enzymatic biosensors exploit the catalytic property of enzymes, biorecognition molecules that possess high chemical specificity and have provided excellent selectivity for their targeted substrate. In enzymatic sensing, the device is combined with a transducer, which reacts selectively with its analyte, generating an electrochemical [54] , optical [55] or piezoelectric [56] signal. This signal correlates to the concentration of the analyte in the sample [57] [58] [59] . Since enzyme electrodes devices offer several distinct advantages, they are used in many point-of-care and clinical applications for a broad range of analytes [60] [61] [62] [63] . Deoxyribonucleic acid (DNA) biosensors, or genosensors, have been exploited for their inherent physicochemical stability and suitability to provide practical ways to identify and diagnose various diseases. DNA is the carrier of genetic information, and it is distinct in any living organism, virus, or pathogen. Therefore, through their specific nucleic acid sequences, the DNA biosensor can be able to discriminate different organisms and diagnose various diseases and human pathogens. The principle of detection of a DNA biosensor relies on the immobilization of an immobilized DNA or RNA strand (probe), on the surface of a physical transducer, to detect its complementary (target) sequence via hybridization. The duplex formation can be detected following the association of an appropriate hybridization indicator, or through other changes accrued from the binding event [64] [65] [66] . Through the efforts of researchers, many new genosensors have emerged in recent years into clinical applications to detect various diseases and pathogens [67] [68] [69] [70] . Whole cells can be used as recognition elements. A variety of surface antigens presented on the cell envelopes, including proteins, glycoproteins, lipopolysaccharides, and peptidoglycan, can act as targets for biorecognition. The development of biosensors for whole microorganisms is challenging because it requires the detection of analytes that are much larger (micrometer scale) than typical molecular analytes, such as proteins (nanometer scale). Many surface epitopes can lead to nonspecific interactions with the sensor surface. Nevertheless, organisms used to develop whole-cell biosensors are generally experimentally modified to incorporate transducer capacity or increase their sensitivity [71] [72] [73] [74] [75] . The whole cell-based biosensor is increasingly being reported in the literature, and these reports have shown high selectivity, sensitivity, and great potential for their use in biomedical diagnostics [76] [77] [78] [79] . As intracellular parasites, viruses use the cellular machineries for completion of their replication cycle. The basic structure of virus is comprised of genome (DNA or RNA), protein capsid (for nucleic acid protection) and in some a lipidic envelope that covers the capsid. [80, 81] . The first reported studies on viruses began at the end of the XIX and the beginning of the XX century, through experiments with tobacco mosaic, in 1882, and with the action of bacteriophages in Shigella culture, in 1915 [82, 83] . The first Influenza virus (FLU) isolated in laboratory was reported in 1933 [84] . It is a respiratory virus, associated with the 1918-1919 pandemic Spanish FLU that resulted in the death of about a quarter of the entire population, and is still the cause of seasonal flu in several countries [85] . From this and previous works, several types of researches on cell culture during the 40s and 50s alongside with the bacteriophage studies of Hershey and Chase in 1952, increased the interest regarding viruses, culminating at the beginning of the modern virology [86] [87] [88] . A virus replication only occurs in intracellular space, a crucial step for virus cycle. Fig.2 represents a generic enveloped RNA virus cycle, the most common among respiratory viruses. In general, the replication steps consist firstly in the attachment between a viral protein and the receptor of the target-cell surface. After the attachment, the virus penetrates the membrane cell to cytoplasm. Right after, an uncoating step is responsible for the release of viral nucleic acid into the host cell and allows the synthesis of viral proteins and genome. Then, the viral particles are assembled according to the viral symmetry. With the new viral particles assembled, the maturation step leaves the virus infectious, increasing the viral tropism. Finally, the virus is released by different mechanisms, like cell lysis, budding, or exocytosis [89, 90] . In upper and lower airways in the respiratory tract, there are physical barriers composed by epithelial cells and mucus besides the alveolar macrophages in the lungs [91] . This set of protection seeks to safeguard the immune response of the host body in several ways, like the inhibition of interferon, latent infections, or the presence of fusion proteins in the viral capsid [90] . Besides, viruses can mutate and might result in more tropism and virulence, thus increasing the evasion from the immunologic system, aggravating the clinical condition of the host [92, 93] . The most worrying respiratory viruses in public health: respiratory syncytial virus (RSV), coronavirus (MERS and SARS-CoV), and FLU [2] , are based in RNA, in which the mutation process is more accentuated [94] . This mutation comes from the low fidelity of RNA polymerase, resulting in the incorporation of nucleotides errors during the replication of the material genetic with more frequency (10 -3 to 10 -4 nucleotides) compared to DNA polymerase (10 -8 to 10 -11 nucleotides), which is present in eukaryotic cells and some classes of viruses, as adenoviruses [94] [95] [96] [97] . The RNA viruses are responsible for countless cases of illness from these acute respiratory tract infections resulting in deaths worldwide, as summarized in Table 1. INSERT TABLE 1 Despite the shorter evaluated period, SARS and MERS shows a high mortality rate. With annual estimates, unfortunately, Influenza and RSV are still the deadliest among the respiratory viruses. The other viruses, although data are not shown in the table, can also lead to death, even in a small proportion or in a local scenario, as it is the case for some reports regarding human adenovirus [98] , human metapneumovirus [99] and rhinovirus [100] . The gold standard test to diagnose respiratory virus disease is based on cell culture, upon the evaluation of the cytopathic effect and hemadsorption caused by the virus [101] . However, molecular assays based on nucleic acid have also been employed [102] [103] [104] [105] [106] as the respiratory virus multiplex PCR systems for respiratory virus detection, with high accuracy for early diagnosis [107] . However, the need for a diagnosis with high accuracy and precision, associated with fast analysis, low cost, in an easy-to-handle device, is still not observed [108, 109] . Therefore, alternatives as the use of biosensors have been under evaluation to supply these demands [110, 111] . The next topics surveyed the most recent contributions related to biosensors applied to the determination of respiratory viruses. This compilation is important to disseminate information to researches interested in the development of such devices. Influenza virus is a member of the Orthomyxoviridae family, with (-)ssRNA nucleic acid, and the species A and B are the most common associated with human infection and disease [112] . The Influenza virus exhaust path consists of the alteration in the hemagglutinin antigens (1-18) and the neuraminidase (1-11), responsible for the attachment and penetration in the host cell, respectively [113, 114] . Upon that, it is possible to distinguish the virus A subtypes. As for the seasonal specie B, it differs basically in the HA1 antigen between the two strains, B/Yamagata/16/88, and B/Victoria/2/87, and the classification is based on this difference [115, 116] . The Influenza virus is responsible for countless deaths around the world, especially at the end of the pandemic in 1910, and only in the 30s, researchers acquired useful information regarding the virus. It is estimated that the Influenza virus, especially the subtype H1N1, has infected about 500 million people [113] . The virus isolation at 1933 [84] , alongside with Louis Pasteur research on a vaccine against rabies, in 1885 [117] , were important developments for the soviet development of the first known attenuated vaccine against the influenza virus A [118] . However, only in the 40s, an inactivated vaccine was approved. Although safer, it was less efficient [81] . It contained viral particles from both Influenza A and B, for population distribution [119] . The majority of biosensor development found in the literature are designed to Influenza virus detection, as attested by the high number of reviews regarding this subject [120] [121] [122] [123] [124] . Table 2 contains data based on biosensor research directed to Influenza virus detection in the past five years. It is the most studied virus among the other respiratory viruses, and some reports stand out for low limit of detection, simplicity, fastness, and cutting-edge technology. In a fast analysis, high sensitivity and selectivity are mandatory. Veerapandian [125] developed an electrochemical biosensor using a carbon screen-printed electrode modified with graphene oxide nanosheets followed by methylene blue adsorption, chitosan, protein-A from S. aureus, and monoclonal antibodies (H5N1 and H1N1) immobilized through drop-casting. Despite the many steps, a simple device can be assembled, with the possibility to detect two different subtypes simultaneously with a limit of detection below nM (9.4 pM for H1N1 and 8.3 for H5N1). The authors also informed the fastness of the analysis, with the detection time below 1 minute, far better characteristics than the traditional methods. This work presents a biosensor with all the desirable features required for this device as high sensitivity, specificity, low cost, fastness, besides the simultaneous determination of two different virus subtypes in one measurement using small-volume samples (50 to 100 μL). Regardless of their classification, antibodies present a unique and specific interaction with the correspondent antigen with a low equilibrium dissociation constant. The use of immunosensors stands out in numbers for Influenza virus detection, as can be seen in Table 2 , including the simultaneous determination of different subtypes. An attractive aspect of the immunosensor is the possibility of the development of two different devices for the same purpose. It is possible to immobilize either the antibody or the antigen. Han [126] developed a biosensor for simultaneous detection of H1N1, H5N1, and H7N9 virus using a triple-arrayed three-electrode chip over a polydimethylsiloxane (PDMS) with a limit of detection as low as 1 pg.mL -1 . In their work, the antibodies were immobilized over the transducer, and the virus antigens were detected. Fig.3 depicts the sensor used by Han. On the other hand, Miluka [127] immobilized the antigen, recombinant Histagged HA subtype H1N1-monomer from the H1N1 pdm09 influenza virus for the detection of anti-hemagglutinin antibodies against the swine virus H1N1. The antigen was immobilized over a dipyrromethene-Cu(II)-modified gold electrode, and the antihemagglutinin H1 antibodies from mice sera were determined. Wong [128] reported a biosensor for H5N1 based on the H5N1 virus protein-modified gold electrode for the detection of H5N1 antibodies. The authors of these two works claimed that their devices present a lower limit of detection than commercial systems. Another type of sensor growing considered attention is the genosensor, also applied to Influenza virus detection. An advantage of using RNA or DNA is that the classic biosensor assembly is dependent on the hybridization of single strands, a reversible process, meaning that the transducer surface can be regenerated. Genosensors are also known for their excellent limit of detection. Ravina [129] reported a biosensor based on a gold screen-printed electrode modified with 5'-amine labeled 22-mer ssDNA of hemagglutinin gene to the detection of the complementary DNA strand through electrochemical impedance spectroscopy (EIS). A limit of detection of 4 pg was obtained. Dong [130] developed an electrochemical genosensor for H7N9 with a limit of detection of 0.75 fM. However, Medina-Sánchez [131] has found an outstanding limit of detection of 20 aM through EIS without any amplification, introducing a new electrode with tubular geometry. Electrochemical transducers are the basis of most of the devices studied for Influenza virus determination, but optical transducers also provide high efficiency and sensitivity. He [132] has found a remarkable limit of detection of 0.53 copies.mL -1 for H5N1 detection in a 20 min experiment using a colorimetric transducer based on the chromism and fluorescence properties of polydiacetylene vesicles modified through covalent binding to HA of H5 monoclonal antibody. The variety of different electrodes, transducers, and detection techniques for Influenza virus detection, encouraged the development of innovative systems. Jang was the first to develop a low-cost, miniaturized paper-based [133] , followed by a flowbased paper [134] immunosensor for electrochemical and colorimetric detection of Influenza H1N1 virus. The evolution from simple paper to flow-paper promoted a decrease in the limit of detection from 113 PFU.mL -1 to 3.3 PFU.mL -1 (electrochemical) and 1.34 PFU.mL -1 (colorimetric). Hushegyi [135] developed a glycan biosensor for the Influenza virus. Glycan are complex organic structures present in the surface of viruses and host cells, as for Influenza viruses and human cells [136] . Tkac used a polycrystalline gold electrode modified with a mixture of two thiols in a self-assembled monolayer (SAM) to immobilize a glycan (a 2,3-sialyllactose derivative). This biosensor was used to detect two Influenza virus subtypes, H1N1 and H5N1, through the interaction between the immobilized glycan and the virus glycoprotein hemagglutinin. This was the first detection at aM range (140 aM for H5N1 and 14 fM for H1N1) using an impedimetric glycan biosensor. A similar approach was used by glycoprotein modified-polyacrylamide. The striking difference is the detection methodologies. In the first moment, they used a standard cantilever from atomic force microscope as the transducer. The measurement was based on the cantilever deflection induced by lateral intermolecular forces in a flow system upon contact with the virus. As it used a complex optical system, it was not classified by the authors for home appliances. Therefore, in the second moment, they switched into a piezoelectric transducer using a lead zirconate titanate piezoelectric disc. By keeping the flowing system and the same syalylglycopolymer, they achieved a sensitive, selective, label-free device that can be applied for commercial use. Real biological samples are usually based on blood, but fluids as urine and saliva can also be used. Another advantage of biosensors for Influenza virus detection is the possibility of using less invasive fluids obtained from mouth, throat, or nose to get viral biological material. Nidworski [139] developed an impedimetric immunosensor for the Influenza virus based on a boron-doped diamond (BDD) electrode functionalized with polyclonal anti-M1 antibodies. Real samples based on throat and nasal swabs were treated to release the M1 protein from the virus. The sensor showed high sensitivity, selectivity, and rapid analysis (5 minutes). The first studies of the coronavirus were performed in the late 1960s by distinct researcher groups. They have isolated, independently, different strains of a new virus in cell culture, which presented an unusual ether-sensitive property. Tyrrell demonstrated through electron microscopy that this new group of viruses was also morphological identical, with a crown-like appearance (so the name corona). Years after the understanding of the first coronavirus strains -B814, 229E, and OC43, and due to the virus study and its presence in many animals, the coronavirus was divided into three distinct groups: the first one containing 229E; the second one containing OC43; and the third one containing aviary virus, all with classification based on the genome and specific antigens [167] . In this century, three significant outbreaks have already changed the world scenario regarding the Coronaviridae family. At the end of 2002 and the beginning of 2003, in southern China, a new type of coronavirus was discovered. Named severe acute respiratory syndrome coronavirus (SARS-CoV), it promoted an outbreak with near a thousand deaths and more than eight thousand infected [168] . In 2012, a second outbreak was attributed to the middle east respiratory syndrome coronavirus (MERS-CoV) [169] , with origin in the south of Asia and the middle east of Africa. Around 35 % of the patients died, a higher mortality than SARS. At the end of 2019, the new pandemic spread worldwide from China was due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19 disease. These viruses' transmission to humans is believed to occur via animals as natural virus reservoirs, especially bats, civets, and camels [168] . The human contact with the animal environment, their secretions, and their meat may be the principal transmission mode. The reason for the high infection rate reported for SARS-CoV-2 is because the virus is easily transmitted via respiratory droplet, through aerial droplets and contact. SARS-CoV-2 has a higher propagation rate when compared to other coronaviruses. It is believed that it has a high viral load right in the beginning of the infection, which can lead to an inter-human transmission ever since. In the other hand, SARS-CoV has different moments of high viral load, higher at the end of the disease, which justify its lower transmission in the beginning of the infection [170] . It is considered that humans are transient or terminal host regarding inter-human infection by the MERS virus, and there are, until now, no supporting evidence that the transmission can occur [171] . The coronaviruses are developed in the host cellular cytoplasm, promoting cell destruction [167] . The diseases can be asymptomatic, specially MERS. Some unspecific symptoms are fever, cough, loss of air, and, in severe cases, severe respiratory problems such as respiratory insufficiency and associated comorbidities [169, 172] . Despite the lethality of SARS-CoV, SARS-CoV-2, and MERS, it was not found in the literature many publications regarding coronavirus biosensor development compared to the growing number of studies for the Influenza virus. A partial explanation may come from the fact that Influenza virus has many harmful subtypes, and the morbidity and mortality are reported annually worldwide, which demands the fast diagnosis with reliability found in biosensors. The more significant events related to coronaviruses were the SARS-CoV and MERS outbreaks, which were controlled. Therefore, fewer efforts are found regarding these two specifics strains. However, the emergence of the new SARS-CoV-2 enhanced the concern, and the research for coronavirus became vital. Within months it was published two papers reporting biosensor development for detection of SARS-CoV-2, so far. These are the first steps for a real device that can be used to identify infected people and hasten the treatment. Table 3 contains data of these two papers alongside other reports regarding SARS, and MERS since the first outbreak, in 2002. It was observed for the biosensors developed for the Influenza virus that electrochemical systems were the majority among the papers found. For coronavirus detection, only six optical systems were found as the main sensor type with one work using a piezoelectric system for SARS-CoV-1 determination, one work using an electrochemical system for MERS determination and one electrochemical system for SARS-CoV-2 determination. The efficient and innovative devices developed for the Influenza virus and presented in this paper can be used as models to the determination of these viruses, since the biological material are essentially the same, such as antibodies, antigens, DNA, PCR products, and more. protein, a specific SARS-CoV antigen as a faster and more sensitive alternative to the RT-qPCR and serological tests. The same limit of detection (0.1 pg.mL -1 ) was also achieved two years after by Roh [174] using quantum dots-conjugated RNA aptamer immobilized over a designed chip to recognize the same N protein from SARS-CoV, and it is, to date, the lowest limit of detection found for a coronavirus through biosensor devices. Seeking an efficient biomolecule immobilization, Park [175] developed a surface plasmon-ressonance (SPR) biosensor for SARS-CoV based on the use of gold binding polypeptide (GBP). GBP was fused to enhanced green fluorescent protein (GBP-E) and to SARS-CoV membrane envelope (SCVme), the latter that can bind to anti-SCVme antibodies. This interesting system presents high specificity to gold substrates without losing the biomolecule activity. A representation of the work of Park can be seen in Fig.4 . All MERS biosensors found were developed as nanoparticle-based devices. An explanation may be that by the time of MERS outbreak, the use of nanoparticles and quantum dots for the most diverse systems were sedimented in the scientific community with easiness in the fabrication, stability, and many nanostructure options. Teengam [176] presented a biosensor for MERS detection as an innovative optical system. It was used a pyrrolidinyl peptide nucleic acid (PNA) immobilized over a paper-based analytical device (PAD) to detect synthetic oligonucleotides with a sequence corresponding to MERS. The paper-type colorimetric biosensor response occurs through aggregation/de-aggregation of negatively charged silver nanoparticles upon addition with positively charged PNA followed by complementary DNA. The results analysis does not require a computer interface, as the responses can be observed in the naked eye. Layqah has recently [177] As far as we know, Qiu and coworkers [178] The biosensors developed for coronavirus presented excellent sensitivity, selectivity, and innovative assembly by taking advantage of the nanotechnology. The recent COVID-19 pandemic already boosted the development of the first biologic sensors, which can imply in revisiting the study of older coronaviruses as SARS and MERS. It is expected more research on these viruses, as simultaneous determination, miniaturization, and development of point-of-care devices for the population. The respiratory syncytial virus (RSV) was firstly isolated from chimpanzees in 1956 [182] . It presents an (-)ssRNA genome [183] and belongs to the Paramyxoviridae family, which is composed of other viruses of public health interest, as the measles virus and the Newcastle disease virus [184] . It is one of the major causes of respiratory diseases around the world with a lethality compared to the Influenza virus, mainly in children under five years old [185] . In this group of children RSV can lead to bronchiolitis and pneumonia, the former presenting in almost 50 % of the cases [182, 186] . There is currently no vaccine against RSV [183] . An attempt was performed in 1967 to develop an inactive vaccine, but it was not effective [187] . This current scenario was responsible for more than 59,000 deaths of children under 5 years old in 2015 [188] with estimative of deaths between 66,000 and 234,000 children, teenagers, and elders [187] . Just in Brazil, RSV was responsible for 2,366 new cases from 2007-2012 with more deaths even than the deaths correlated with the Influenza virus [185] . It is interesting to evaluate the discrepancies in the biosensor developments in the past few years. There are currently only vaccines for some Influenza virus subtypes, but none for the coronaviruses presented here and RSV. Therefore, the previous determination of these viruses in the contaminated people is of utmost importance for healthcare and to prevent new epidemics or even worse as SARS-CoV-2 pandemic. However, even with all of these concerns, few studies were reported with the development of biosensors for RSV detection. Table 4 contains the data of the few papers found in the literature. Only three optical systems and two electrochemical systems were found. Nevertheless, regarding the other viruses approaches, RSV biosensors present more heterogenicity than the others. With the purpose to detect viral particles, Peres [189] developed an optic genosensor based in gold filament substrate. With a detection limit of 11.9 PFU, this is one of the most sensitive biosensors for RSV detection. Rochelet [190] developed an immunosensor based on a polymer-modified screen-printed electrode. With a low limit of detection of 1:5000 RSV Ag dilution, the device showed efficiency compared to a standard serological assay. Moreover, the device also presented low cost, and fast response time (25 minutes), features not related to the ELISA or the RT-PCR assays. Cai [191] developed a genosensor based on an interesting allosteric molecular beacon (aMB), as depicted in the Fig.5 . The aMB used in their work, upon contact with an RSV specific DNA target, has its stem opened and formed a streptavidin aptamer that was coupled to a streptavidin-horseradish peroxidase enzyme (SA-HRP). The electrochemical reduction signal of 3,3',5,5'-tetramethylbenzidine (TMB) formed by the enzymatic reaction in the hydrogen peroxide presence was correlated to RSV concentration. With a limit of detection of 11.0 pM using a small sample volume (4.0 μL), this biosensor presented the lowest LOD among the studies found for RSV detection. The aMB strategy provides excellent sensitivity, which was also observed for the Influenza virus [192] , and it is a new alternative for genosensor development. Shi [181] developed a genosensor based on gene gold chips through surface plasmon resonance that was able to detect simultaneously nine respiratory viruses: influenza virus A and B, H1N1, RSV, parainfluenza virus 1-3 (PIV1, 2, 3), adenovirus, and SARS-CoV-1. The biosensor presents high sensitivity and selectivity. Moreover, it represents an important step for further studies in the simultaneous determination of several viruses, since the associated illnesses cause similar symptoms. The possibility to discard and to confirm diseases with reliability is the ultimate goal in the frontline in the fight against respiratory viruses. Despite the high incidence rates and mortality related to the viruses previously described in this paper, other respiratory viruses present lower virulence, but they are responsible for complications in humans and overload in health systems, like human adenovirus (AdV), human bocavirus (HBoV) and human Rhinovirus (HRV) [194] [195] [196] . Probably because of their low virulence, there are just a few biosensors reporting their determination. Regardless, the development of such device is of the most importance because the symptoms that these viruses cause are the same of the other viruses reported so far. The use of these specific biosensors by the population may be the first step to rule out other deadly viruses and, thus, to decrease the swelling in health services. From the few papers found, it is worth mention the work of Ostroff [197] , the only biosensor developed for human rhinovirus (HRV) determination, using an optically coated silicon surface modified with HRV polyclonal antibodies. The system was able to detect the virus-specific non-structural protein 3C protease. The biosensor presented high sensitivity (picomolar range) and fast response 28 minutes. One significant advantage of biosensors is the possibility of simultaneous determinations of several analytes. Alongside the many requirements for a biosensor, the development of a device that could analyze and discriminate several respiratory viruses in one single analysis is a remarkable achievement. The works of Jin [198] , Shi [181] , and Jenison [199] were performed regarding human adenovirus. The work of Shi was already discussed for RSV because this device was able to determine nine different respiratory viruses, including human adenovirus. Jin developed an electrochemical biosensor based on gold chips modified with carbon nanotubes to determine the virus through surface plasmon resonance. The work of Jenison went through the same way, with a ten-minute assay for the detection of seven respiratory viruses, including rhinovirus, as depicted in Fig.6 . A great number of papers are addressed to the development of biosensors, but still, few devices are available in commerce as the glucose biosensor or using commercial detection systems. One good example of respiratory virus detection is the work of Owens [200] , in which it was developed a label-free optical biosensor for the determination of HRV using the Corning Epic® system, as depicted in Fig.7 . This work, alongside other studies presented here, shows an excellent perspective for commercial devices. There are other important respiratory viruses, but it was not found any biosensors reports, such as the human bocavirus, which proves that much work can still be performed to achieve early diagnosis and availability of commercial devices for these viruses determination [194] . The diseases caused by respiratory viruses are a matter of public health, with more The use of commercial electrodes such as screen-printed, and systems that can detect more than one respiratory virus in a simultaneous determination, provide some perspective. There is not available any device, as the glucose biosensor, regarding the determination of the viruses reported in this review. Nevertheless, with just months since the beginning of the COVID-19 pandemic, at least two biosensors were developed for SARS-CoV-2 determination, which can be the first step for further studies to evolve from the proof of concept to the point of care device. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. World Health Organization (WHO) World Health Organization (WHO), WHO Director-General's opening remarks at the media briefing on COVID-19 -16 Detection of 2019 novel coronavirus ( 2019-nCoV ) by real-time RT-PCR Evaluation of Enzyme-Linked Immunoassay and Colloidal Gold-Immunochromatographic Assay Kit for Detection of Novel Coronavirus ( SARS-Cov-2 ) Causing an Outbreak of Pneumonia ( COVID-19 ) Biosensor Regeneration: A Review of Common Techniques and Outcomes Transduction Process-Based Classification of Biosensors Fabricating Long Shelf Life Potentiometric Urea Biosensors Using Modified MWCNTs on Screen Printed Electrodes Recent advances in potentiometric biosensors Amperometric Glucose Biosensor Based on Electrochemically Deposited Gold Nanoparticles Covered by Polypyrrole MON-182 Adaptable Amperometric Biosensor Platforms for the Diagnosis of Endocrine Disorders Selective Determination of Verapamil in Pharmaceutics and Urine Using a Boron-doped Diamond Electrode Coupled to Flow Injection Analysis with Multiple-pulse Amperometric Detection Highly sensitive impedimetric immunosensor for determination of interleukin 6 as a cancer biomarker by using conjugated polymer containing epoxy side groups modified disposable ITO electrode Impedance-based biosensors Label-free electrochemical impedance immunosensor based on modified screen-printed gold electrodes for the diagnosis of canine visceral leishmaniasis Smartphone based optical biosensor for the detection of urea in saliva Surface Plasmon Resonance Clinical Biosensors for Medical Diagnostics Boosting Cancer Immunotherapies with Optical Biosensor Nanotechnologies Biosensors Approach for Lung Cancer Diagnosis-A Review Piezoelectric biosensor for the determination of Tumor Necrosis Factor Alpha A review of experimental aspects of electrochemical immunosensors Molecular Imprinting of Macromolecules for Sensor Applications Immunosensors in Clinical Laboratory Diagnostics Advanced surface modification technologies for biosensors The Potential of a Nanostructured Titanium Oxide Layer with Self-Assembled Monolayers for Biomedical Applications: Surface Properties and Biomechanical Behaviors Rodríguez-Cabello, Recombinant AMP/Polypeptide Self-Assembled Monolayers with Synergistic Antimicrobial Properties for Bacterial Strains of Medical Relevance Introduction to medical biosensors for point of care applications Nanostructured materials and nanoparticles for point of care (POC) medical biosensors Coronavirus COVID-19 impacts to dentistry and potential salivary diagnosis Novel Coronavirus Disease (COVID-19): Paving the Road for Rapid Detection and Point-of-Care Diagnostics Cellular and molecular immunology Applications of electrochemical immunosensors for early clinical diagnostics Ultrasensitive amperometric immunosensor for PSA detection based on Cu2O@CeO2-Au nanocomposites as integrated triple signal amplification strategy A label-free electrochemical immunosensor for the detection of cardiac marker using graphene quantum dots Gold nanoparticle amplification strategies for multiplex SPRi-based immunosensing of human pancreatic islet hormones Impedimetric and electrochemical evaluation of a new redox active steroid derivative for hormone immunosensing Diazonium-Modified Screen-Printed Electrodes for Immunosensing Growth Hormone in Blood Samples Electrochemical immunosensor based on gold-labeled monoclonal anti-LipL32 for leptospirosis diagnosis Immunosensing system for rapid multiplex detection of mastitis-causing pathogens in milk Highly sensitive electrochemical immunosensor based on graphene-wrapped copper oxide-cysteine hierarchical structure for detection of pathogenic bacteria Development of a rapid and sensitive immunosensor for the detection of bacteria Electropolymerization of hydroxyphenylacetic acid isomers and the development of a bioelectrode for the diagnosis of bacterial meningitis A promising magnetic SERS immunosensor for sensitive detection of avian influenza virus Immunosensor-based label-free and multiplex detection of influenza viruses: State of the art A new tool for dengue virus diagnosis: Optimization and detection of anti-NS1 antibodies in serum samples by impedimetric transducers A sensitive electrochemical immunosensor for label-free detection of Zika-virus protein Rapid and Sensitive Nano-Immunosensors for Botulinum Electrospun Carbon Nanofibers as an Electrochemical Immunosensing Platform for Vibrio cholerae Toxin: Aging Effect of the Redox Probe Dual-channel ITO-microfluidic electrochemical immunosensor for simultaneous detection of two mycotoxins Bismuth oxide nanorods based immunosensor for mycotoxin detection Sensitive detection of maltose and glucose based on dual enzyme-displayed bacteria electrochemical biosensor An optical biosensor for the determination of cathepsin B as a cancer-associated enzyme using nanoporous anodic alumina modified with human serum albumin-thionine Selfpowered gustation electronic skin for mimicking taste buds based on piezoelectric-enzymatic reaction coupling process Enzyme based amperometric biosensors Biosensors and their applications -A review Fundamentals of Enzyme Engineering, 1st Edit A numerical modelling of an amperometric-enzymatic based uric acid biosensor for GOUT arthritis diseases A portable microfluidic Aptamer-Tethered Enzyme Capture (APTEC) biosensor for malaria diagnosis Recent Advances in Electrochemical Biosensors Based on Enzyme Inhibition for Clinical and Pharmaceutical Applications An enzyme-free electrochemical biosensor based on well monodisperse Au nanorods for ultra-sensitive detection of telomerase activity Electrochemical DNA sensors based on the use of gold nanoparticles: a review on recent developments The strategies of DNA immobilization and hybridization detection mechanism in the construction of electrochemical DNA sensor: A review Detection of Neisseria meningitidis using surface plasmon resonance based DNA biosensor DNA biosensors based on gold nanoparticles-modified graphene oxide for the detection of breast cancer biomarkers for early diagnosis A liquid-crystal-based DNA biosensor for pathogen detection A label-free electrochemical DNA biosensor for breast cancer marker BRCA1 based on self-assembled antifouling peptide monolayer Biosensors for Whole-Cell Bacterial Detection A review of impedance measurements of whole cells Miniaturized and integrated whole cell living bacterial sensors in field applicable autonomous devices Bacterial host and reporter gene optimization for genetically encoded whole cell biosensors The Application of Whole Cell-Based Biosensors for Use in Environmental Analysis and in Medical Diagnostics A label-free optical whole-cell Escherichia coli biosensor for the detection of pyrethroid insecticide exposure Whole-Cell Biosensor with Tunable Limit of Detection Enables Low-Cost Agglutination Assays for Medical Diagnostic Applications Cell-based biosensors for immunology, inflammation, and allergy Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production The Origins of Viruses Vírus, Viróides e Prions Tobacco Mosaic Virus Epidemiology and Control Félix Hubert d'Herelle (1873-1949): History of a scientific mind Pandora' s Box and the History of the Respiratory Viruses : A Case Study of Serendipity in Research Reviewing the History of Pandemic Influenza: Understanding Patterns of Emergence and Transmission Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage Cultivation of the Lansing Strain of Poliomyelitis Virus in Cultures of Various Human Embryonic Tissues History and Impact of Virology Virus Replication Respir. Dis. Infect. -A New Insight, First Edit, IntechOpen The mucosal immune system of the respiratory tract PB2 and HA mutations increase the virulence of highly pathogenic H5N5 clade 2.3.4.4 avian influenza virus in mice Coronavirus Spike Protein and Tropism Changes Princ. Mol. Virol., 5th Editio Expression of Animal Virus Genomes World Health Organization (WHO), Viruses, in: Water Recreat Fenner White's Med. Virol., 5th Edit Fatalities Associated with Human Adenovirus Type 7 at a Substance Abuse Rehabilitation Facility -New Jersey Human Metapneumovirus: Lessons Learned over the First Decade Unexpectedly Higher Morbidity and Mortality of Hospitalized Elderly Patients Associated with Rhinovirus Compared with Influenza Virus Respiratory Tract Infection Laboratory Diagnosis of Respiratory Tract Infections in Children -the State of the Art Detection of respiratory viruses by real-time polymerase chain reaction in outpatients with acute respiratory infection Detection of Respiratory Viruses by Molecular Methods, Clin PCR for detection of respiratory viruses: seasonal variations of virus infections Increased Detection of Viruses in Children with Respiratory Tract Infection Using PCR Making Sense of Respiratory Viral Panel Results Multiplex PCR system for the rapid diagnosis of respiratory virus infection : systematic review and meta-analysis Biosensors for clinical diagnostics industry Disposable Biosensors for Clinical Diagnosis The role of biosensors in the detection of emerging infectious diseases The Editors of Encyclopaedia Britannica Vaccine-Preventable Dis., 13th Edit, Public Health Foundation Biosensor for Rapid and Sensitive Detection of Influenza Virus Cocirculation of two distinct evolutionary lineages of influenza type B virus since 1983 Types of Influenza Viruses, CDC Inner Workings: 1885, the first rabies vaccination in humans Influenza Virus Vaccines: Past and Future The Evolving History of Influenza Viruses and Influenza Vaccines DFT theoretical study of 7-R-3methylquinoxalin-2(1H)-thiones Cl) as corrosion inhibitors in hydrochloric acid Detection Methods of Human and Animal Influenza Virus-Current Trends Miniaturized biosensor for avian influenza virus detection Detection methods for influenza A H1N1 virus with special reference to biosensors: a review Biosensor for Rapid and Sensitive Detection of Influenza Virus Dual immunosensor based on methylene blue-electroadsorbed graphene oxide for rapid detection of the in fl uenza A virus antigen A multi-virus detectable microfluidic electrochemical immunosensor for simultaneous detection of H1N1, H5N1, and H7N9 virus using ZnO nanorods for sensitivity enhancement Highly sensitive electrochemical biosensor based on redox -active monolayer for detection of anti-hemagglutinin antibodies against swine-origin influenza virus H1N1 in sera of vaccinated mice A Phase-Intensity Surface Plasmon Resonance Biosensor for Avian Influenza A (H5N1) Hemagglutinin gene based biosensor for early detection of swine flu (H1N1) infection in human Electrochemical DNA Biosensor Based on a Tetrahedral Nanostructure Probe for the Detection of Avian Influenza A (H7N9) Virus High-performance 3D tubular nanomembrane sensor for DNA detection Development and evaluation of a polydiacetylene based biosensor for the detection of H5 influenza virus Cost-Effective and Handmade Paper-Based Immunosensing Device for Electrochemical Detection of Influenza Virus Vertical flow-based paper immunosensor for rapid electrochemical and colorimetric detection of influenza virus using a different pore size sample pad Ultrasensitive detection of influenza viruses with a glycan-based impedimetric biosensor Glycan-protein interactions in viral pathogenesis Synthetic sialylglycopolymer receptor for virus detection using cantilever-based sensors Label-free sensitive detection of influenza virus using PZT discs with a synthetic sialylglycopolymer receptor layer A rapid-response ultrasensitive biosensor for influenza virus detection using antibody modified boron-doped diamond Low cost synthesis of reduced graphene oxide using biopolymer for influenza virus sensor Electrochemical DNA Sensor for Simultaneously Detecting and Subtyping Influenza A Viruses Subtyping of influenza A H1N1 virus using a label-free electrochemical biosensor based on the DNA aptamer targeting the stem region of HA protein Detection of influenza virus by a biosensor based on the method combining electrochemiluminescence on binary SAMs modified Au electrode with an immunoliposome encapsulating Ru ( II ) complex Label-free Detection of Influenza Viruses using a Reduced Graphene Oxide-based Electrochemical Immunosensor Integrated with a Microfluidic Platform Simple Strategy for Rapid and Sensitive Detection of Avian Influenza A H7N9 Virus Based on Intensity-Modulated SPR Biosensor and New Generated Antibody Amperometric bioaffinity sensing platform for avian influenza virus proteins with aptamer modified gold nanoparticles on carbon chips Fabrication of electrochemical biosensor consisted of multi functional DNA structure/porous au nanoparticle for avian influenza virus (H5N1) in chicken serum Highly sensitive colorimetric immunosensor for influenza virus H5N1 based on enzyme-encapsulated liposome Highly sensitive sandwich-type SPR based detection of whole H5Nx viruses using a pair of aptamers A biosensor based on electroactive dipyrromethene-Cu(II) layer deposited onto gold electrodes for the detection of antibodies against avian influenza virus type H5N1 in hen sera Ultrasensitive electrochemical genosensor for direct detection of specific RNA sequences derived from avian influenza viruses present in biological samples A fluorescent aptasensor for H5N1 influenza virus detection based-on the CORE-shell nanoparticles metalenhanced fluorescence (MEF) Avian Influenza Virus Detection by Optimized Peptide Termination on a Boron-Doped Diamond Electrode Prevention and control of emergent infectious disease with high specific antigen sensor A selfcalibrating electrochemical aptasensing platform: Correcting external interference errors for the reliable and stable detection of avian influenza viruses Fully Packaged Portable Thin Film Biosensor for the Direct Detection of Highly Pathogenic Viruses from On-Site Samples Silver nanoparticles coated graphene electrochemical sensor for the ultrasensitive analysis of avian influenza virus H7 Rapid and background-free detection of avian influenza virus in opaque sample using NIR-to-NIR upconversion nanoparticlebased lateral flow immunoassay platform Giant Magnetoresistance-based Biosensor for Detection of Influenza A Virus Competitive non-SELEX for the selective and rapid enrichment of DNA aptamers and its use in electrochemical aptasensor An Antibody-Immobilized Silica Inverse Opal Nanostructure for Label-Free Optical Biosensors Labelfree localized surface plasmon resonance biosensor composed of multi-functional DNA 3 way junction on hollow Au spike-like nanoparticles (HAuSN) for avian influenza virus detection Facile Hydrothermal Growth Graphene/ZnO Nanocomposite for Development of Enhanced Biosensor Aptamer-Antibody Complementation On Multiwalled Carbon Nanotube-Gold Transduced Dielectrode Surfaces To Detect Pandemic Swine Influenza Virus Optical fiber sensor based on surface plasmon resonance for rapiddetection of avian influenza virus subtype H6: Initial studies Detection of avian influenza virus subtype H5 using a biosensor based on imaging ellipsometry History and Recent Advances in Coronavirus Discovery The battle against SARS and MERS coronaviruses: Reservoirs and Animal Models Middle East respiratory syndrome coronavirus (MERS-CoV) Viral Load in Upper Respiratory Specimens of Infected Patients Middle East Respiratory Syndrome Coronavirus Transmission SARS (Severe Acute Respiratory Syndrome), WHO Detection of severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein in human serum using a localized surface plasmon coupled fluorescence fiber-optic biosensor Quantitative and sensitive detection of SARS coronavirus nucleocapsid protein using quantum dots-conjugated RNA aptamer on chip A Self-Assembled Fusion Protein-Based Surface Plasmon Resonance Biosensor for Rapid Diagnosis of Severe Acute Respiratory Syndrome Multiplex paper-based colorimetric DNA sensor using pyrrolidinyl peptide nucleic acid-induced AgNPs aggregation for detecting MERS-CoV, MTB and HPV oligonucleotides An electrochemical immunosensor for the corona virus associated with the Middle East respiratory syndrome using an array of gold nanoparticle-modified carbon electrodes Dual-Functional Plasmonic Photothermal Biosensors for Highly Accurate Severe Acute Respiratory Syndrome Coronavirus 2 Detection Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor Piezoelectric quartz crystal aptamer biosensor for detection and quantification of SARS CoV helicase protein, in: 17th Int Development of SPR biosensor for simultaneous detection of multiplex respiratory viruses Viral and Host Factors in Human Respiratory Syncytial Virus Pathogenesis Respiratory Syncytial Virus: Virology, Reverse Genetics, and Pathogenesis of Disease Paramyxoviridae and Pneumoviridae Respiratory syncytial virus causes more hospitalizations and deaths in equatorial Brazil than influenza (including during the 2009 pandemic) Current approaches to the development of vaccines effective against parainfluenza and respiratory syncytial viruses Brief History and Characterization of Enhanced Respiratory Syncytial Virus Disease Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study Viral detection using DNA functionalized gold filaments A thin layer-based amperometric enzyme immunoassay for the rapid and sensitive diagnosis of respiratory syncytial virus infections An electrochemical sensor based on label-free functional allosteric molecular beacons for detection target DNA/miRNA Recognition of Dual Targets by a Molecular Beacon-Based Sensor: Subtyping of Influenza A Virus Sensitive immunosensor for respiratory syncytial virus based on dual signal amplification of gold nanopaticle layer-modified plates and catalyzed reporter deposition An enhanced immunoassay was designed and utilized for respiratory syncytial virus ( RSV ) Human Bocavirus: Lessons Learned to Date Respiratory Viruses Other than Influenza Virus: Impact and Therapeutic Advances Impact and seasonality of human rhinovirus infection in hospitalized patients for two consecutive years Rapid multiserotype detection of human rhinoviruses on optically coated silicon surfaces Detection of human adenovirus hexon antigen using carbon nanotube sensors Use of a thin film biosensor for rapid visual detection of PCR products in a multiplex format Real-time quantitation of viral replication and inhibitor potency using a label-free optical biosensor SL-PAA = synthetic sialylglycoconjugates based on a polymer matrix 3WJ = 3 way-junction Ag@SiO 2 NPs = silver@silicon dioxide nanoparticles AIV = Avian influenza viruses APP = 4-amino phenyl phosphate DEP = disposable three-electrode screen-printed DPV = differential pulse voltammetry His6-H5 = histidine-tagged hemagglutinin ellipsometry; IM-SPR = intensity-modulated surface plasmon resonance IPA = intermittent pulse amperometry LSPR = localized surface plasmon resonance LSV = linear sweep voltammetry MOSFET = metal−oxide semiconductor field-effect NNLPA = NIR-to-NIR lateral flow immunoassay OSWV = Osteryoung square-wave voltammetric SiO 2 = silicon dioxide SPCE = screen-printed carbon electrode SPE = screenprinted electrode SPM = scanning probe microscopy SPR = surface plasmon resonance; ss-cDNA = single stranded-cDNA TFT = thin film transistor COVID-19 patients acpcPNA = pyrrolidinyl peptide nucleic acid FET = Field-effect transistor; GBP-E = Gold binding polypeptides-enhanced green fluorescent protein The authors are grateful for the technical support from the Federal [193] aMB = Allosteric molecular beacons; SA-HRP = Streptavidin aptamer-horseradish peroxidase; TMB = 3,3,5,5-Tetramethylbenzidine;