key: cord-0907027-omzhhr0k authors: Kaya, Sariye Irem; Karadurmus, Leyla; Ozcelikay, Goksu; Bakirhan, Nurgul K.; Ozkan, Sibel A. title: Chapter 18 Electrochemical virus detections with nanobiosensors date: 2020-12-31 journal: Nanosensors for Smart Cities DOI: 10.1016/b978-0-12-819870-4.00017-7 sha: bb6aad787f32fe521ff7ab6a80f6c832521bad35 doc_id: 907027 cord_uid: omzhhr0k Abstract Infectious diseases are caused from pathogens, which need a reliable and fast diagnosis. Today, expert personnel and centralized laboratories are needed to afford much time in diagnosing diseases caused from pathogens. Recent progress in electrochemical studies shows that biosensors are very simple, accurate, precise, and cheap at virus detection, for which researchers find great interest in this field. The clinical levels of these pathogens can be easily analyzed with proposed biosensors. Their working principle is based on affinity between antibody and antigen in body fluids. The progress still continues on these biosensors for accurate, rapid, reliable sensors in future. In addition to a template DNA, primers, Taq polymerase, nucleotides, and a thermocycler are necessary for PCR process [13] . PCR is a significant technique for not only measuring viral nucleic acids but also for medical diagnosis, plant industry, environmental studies, and gene therapy [13] . PCR is one of the most commonly used methods for the detection of viral nucleic acids. Using PCR, we can determine the identity and/or quantity of viral genomes of virions and infected cells [12, 14] . PCR, as a sensitive method, allows searching multiple possible viruses in samples and recognizing virus groups through common sequences [14] . Despite all those advantages, it is really hard to prevent patient samples which will be used for diagnostic PCR analysis from contamination and that can be considered a setback [14] . PCR analysis, also known as reverse transcription PCR (RT-PCR), can also be used to identify viral RNA by adding an initial step in which RNA is converted to DNA [15] . Reverse transcription is carried out by using an enzyme called reverse transcriptase [16] . This conversion step is important because it provides obtaining a DNA more stable than RNA and not easily denatured like RNA for PCR process [16] . Immunoblotting: Immunoblotting, also known as Western blotting, is used for identifying proteins and protein on a membrane, and protein concentration changes in samples with antibodies [17] . The immunoblotting technique determines specific viral proteins insulated from a cell, tissue, organ, or body fluid. With immunoblotting method the infection stage of patient can be determined depending on which antibody is developed against which antigen [12] . Although the difficulty and cost of interpreting the results of immunoblotting results in a decrease in the overall use of this technique, immunoblots are still widely used for diagnostic and research purposes [12, 18] . Immunoprecipitation (IP): IP is the method of precipitating a protein antigen out of solution using an antibody that specifically binds to that specific protein. This technique can be used to isolate and concentrate a particular protein from a sample containing thousands of different proteins [19] . It is a helpful method because it provides purification of viruses which cannot be purified with standard methods, and it also allows quick purification of intact virions from small tissues for transmission electron microscopy (TEM) analysis [20] . Nevertheless, virus-specific antibodies are essential for IP and that can be considered a disadvantage [20] . Enzyme-linked immunosorbent assay (ELISA): Immunoassays, the most popular type of which is ELISA, provide quantification and identification of various antigens [21] . ELISAs involve simple enzyme assays with specificity of antibodies using antigens or antibodies linked to an easily assayed enzyme. The basic principle of ELISA technique is radioimmunoassay process that involves a tagged antibody in order to detect antigens [14, 21] . An enzyme bound with a substrate, a fluorescent molecule, or a radioactive isotope can be described as the tag [14] . ELISA has been used in various fields such as environmental studies in order to detect pollutants, food industry in order to detect toxins or allergens, and medical studies in order to detect pharmaceuticals, disease markers [22] . When we approach ELISA in terms of viruses, it can provide the detection of a virus or an antibody from a sample of a patient infected by viruses [14] . The quantity of viruses from a cell culture/sample or a specific antibody to a specific viral pathogen can be detected using ELISA [14] . Those studies are very crucial for diagnostics [22] . ELISA is a much faster method than immunoblotting to detect a specific viral protein. These are considered highly sensitive methods that can detect very low amounts of protein. On the other hand, ELISA tests can sometimes be quite expensive due to the cost of reagents being used [23] . Direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA are the different types of ELISA tests which are used for different scientific fields [14, 21, 22] . Hemagglutination assay (HA): Basically, hemagglutination can be described as the aggregation of red blood cells (RBCs) due to the presence of hemagglutinating agents such as viruses [24] . Some viruses, such as influenza virus, rabies virus, rubella virus, mumps virus, and measles virus, have hemagglutinin surface proteins that attach to surface glycoproteins of RBCs [25] . HA is a method which relies on the fact that many viruses contain proteins that can bind and agglutinate RBCs. The principle of this assay is simple; however, sample preparation is considered laborious and presents some drawbacks [26] . HA is one of the most widely used techniques in order to detect the presence and quantity of viruses in various samples [24, 25] . However, it cannot give information about the level or measure of viral infectivity [24] . HA is also used for detecting the antibody levels against specific viruses in patient samples. The presence of specific antibodies will prevent the hemagglutination of RBCs by binding the viruses [25] . The results of HAs are given as hemagglutination unit (HAU) [27] . In an assay, in various samples, the amount of viruses in the one that can agglutinate all RBCs is described as 1 HAU [27] . Flow cytometry (FCM): FCM is a commonly used technique in biology, microbiology, virology, and immunology [28] . FCM technique involves investigating cells, cell populations, and antigens from human samples such as several body fluids [28, 29] . Nowadays, with current technological improvements, FCM method provides the determination of particles that have sizes ranging from 100 to 1000 nm such as viruses/viral particles [28] . FCM has become an important device in virology, due to its applications in viral replication and viralÀcell interactions, as well as its capacity to quantify proteins [30, 31] . FCM is used for the diagnosis of viral diseases [29] . Transmission electron microscopy: TEM which is a significant method for diagnostic area can be described as a microscopy technique in which electrons pass through various samples and compose an image of the sample [32] . It allows showing very small materials and due to that, it is used in medical sciences, virology, physical sciences, etc. [32, 33] . Viruses are very small, and most of them can only be viewed by TEM. Therefore TEM has made significant contributions to virology such as the discovery of many viruses, diagnosis of various viral infections, and basic research of virusÀhost cell interactions [34] . Despite being a relatively quicker method and providing qualitative and quantitative information about virions, as a disadvantage, this method has a high detection limit and can only be applied to high virus concentrations ($10 7 particles/mL) [33] . Microscopy in cell culture: Cell culture means growing living cells outside of living organisms in controlled conditions [25] . In order to produce a cell culture the most significant thing is doing all the work under sterile conditions [14] . First step of producing a cell culture is removing and mincing the required tissue. After that, different enzymes such as collagenase are applied to the tissue for degradation of extracellular matrix and releasing cells. Then centrifugation step is applied. Later, cells and growth media are combined with culture dishes and contained in an incubator with humidity, 37 C temperature and 5% CO 2 . Lastly, cells grow and divide by linking to the dish [14] . Cell culture-based virus isolation has been accepted as a "gold standard" in the detection and identification of viruses and is the technique by which all other test methods have been compared [35] . Immunofluorescence assay (IFA): IFA is a diagnostic technique that uses the interaction between viruses and specific antibodies [14, 25] . IFA can be considered a relatively quick method, and it is easy to apply without special technicians, it provides the certain identification of the virus [25] . It is one of the commonly used techniques for the rapid detection of virus infections by identifying virus antigens. IFA staining usually gives very rapid, sensitive, and specific virus identification in about 1À2 hours. Unfortunately, the IFA technique may not be able to verify the identity of all virus strains and may be quite expensive due to the cost of the antibodies used [36] . Before the availability of ELISA tests, IFA was used for diagnostics, but now it is only used for research studies [27] . Viral plaque assay: Viral plaque analysis is one of the most commonly used methods in virology to determine viral titer, and this technique is thought to be effective only for viruses that can infect single-celled cells and replicate cells. With this method, it is possible to determine infectious dose; in other words, the quantitative amount of infectious virus particles [27, 37] . The results are expressed as plaque forming units (PFU) [27] . This method usually requires 4À10 days depending on the virus being analyzed and is considered time-consuming [38] . Viral plaque assay results can change depending on the assay conditions and the PFU results that were found may not always show the certain amount of infectious viral particles [37] . Quantal assays-TCID 50 , LD 50 , EID 50 : Procedures, such as TCID50, LD50, and EID50 assays, are used to determine the infectious titer of virus types that can cause cytopathic effects in tissue culture over a period of 5À20 days. Immunofluorescence foci assay: The immunofluorescence foci assay is a quick method of virus titration that allows the measurement of virus in cell lines, which does not promote plaque occurrence or do not exhibit detectable cytopathic effect (CPE) [39] . The purpose of this review is to discuss the methods used to detect viruses and to summarize the studies on electrochemical nanobiosensors. Rapid assessment of pathogenicity and virulence is the key to taking appropriate health measures in outbreaks. The most important precondition for the fight against viruses is the early isolation and detection of the presence of viral nucleic acid. In conventional methods for detecting viruses, equipment and personnel are required, and furthermore, the diagnosis of infection takes longer. Given these challenges, it is important to find rapid methods for detecting the virus in an easy, inexpensive, sensitive, and selective way in the environment, body fluids, and tissues. The use of nanoparticles (NPs) in combination with electrochemical detection is promising in detecting viruses. The biosensor is an analytical device used for the detection of analytes that combine a biological component with a physicochemical detector [40] . A biosensor may be defined as an analytical device comprising a transducer portion and a biological element [41À43] . Compared to conventional techniques, electrochemical nanobiosensors are faster, practical, precise, selective, and economical. The NP-based biosensor has high specificity and can easily be used, having low cost and precision required for rapid and reproducible detection of pathogenic microorganisms in clinical specimens [44À46]. Viral diseases continue to be one of the most important causes of morbidity and premature death in the human population worldwide. In addition, there is a constant threat of the emergence of new viruses that affect us [47, 48] . Viruses that cause major diseases in humans are as follows: Herpesvirus: Herpesviruses are one of the most well-known viruses among all because they can cause infections for a wide variety of animals from birds to humans [49, 50] . The virions of herpesviruses are enveloped, spherical and have double-stranded DNA [51] . Herpesviruses can affect skin, nervous system, genital tract, etc. and cause several wellknown diseases in humans such as chickenpox, genital herpes, and cold sore [49, 50] . Moreover, they are also related with human cancers such as nasopharyngeal carcinoma and lymphoma (EpsteinÀBarr virus; a species of Herpesvirus) [50] . Those are the types of Herpesviruses that cause infections in humans: Rhadinovirus also known as Kaposi's sarcoma herpes virus that infects B lymphocytes and epithelial cells and causes lymphoma and sarcoma; Simplex virus also known as Herpes simplex virus that infects epithelial cells and causes genital and oral herpes; Varicellovirus also known as Varicella-zoster virus that infects epithelial cells and causes chickenpox; Cytomegalovirus that infects epithelial cells, monocytes, endothelial cells and causes congenital defects; Lymphocryptovirus also known as EpsteinÀBarr virus that infects B lymphocytes and epithelial cells and causes Burkitt's lymphoma, Hodgkin's lymphoma, and nasopharyngeal carcinomas [49, 52] . Paramyxovirus: Paramyxovirus virions are enveloped, spherical with viral RNA [53] . They are responsible for wide variety of diseases in human beings ranging from mumps and measles to aseptic meningitis [54, 55] . In addition to this, Canine morbillivirus that is a member of Paramyxoviridae causes canine distemper disease in some animals [54] . Primary viruses from Paramyxoviridae that infects humans are given next: mumps virus is responsible for mumps disease that is characterized by parotitis and can be very serious in the adulthood. Newcastle disease virus is the cause of virulent Newcastle disease which affects avian species but also it can transmit to humans [53] . Measles virus is an extremely contagious virus which is responsible for measles disease that affects skin, respiratory, and immune systems [56] . The virus infects immune and epithelial cells and measles symptoms are often confused with common cold [56] . Nowadays, there are vaccines available against most of the Paramyxoviruses such as measles, mumps, canine distemper, and Newcastle disease [54] . Parvovirus: Parvoviruses are one of the smallest viruses of all with an enveloped and icosahedral virion that has single-stranded DNA [57, 58] . They can affect a wide variety of creatures ranging from humans to mice and mosquitoes [57] . Adeno-associated virus is considered the prototype of parvoviruses, and it infects humans but does not cause any diseases, only mild infections [57] . In other respects, Adeno-associated virus is used as therapeutic gene delivery vector [58] . B19 virus is a parvovirus which is the cause of erythema infectiosum that infects erythrocytes in children [57] . Minute virus of mice infects T lymphocytes of mice and causes very infectious disease especially in laboratory mice [59, 60] . Canine parvovirus is the reason of gastroenteritis which is a fatal disease for dogs [57] . Human bocavirus is a parvovirus that infects humans and causes respiratory infections with mild symptoms, especially in summertime. It was first isolated both in bovine and canine [57] . Retrovirus: Retrovirus virions are enveloped with RNA that plays a role as a template for synthesis of doublestranded DNA and that is the origin of "retro" name of virus [61] . Retroviruses are related with transmissible cancers, for example, HIV [61] . Avian leucosis virus uses chickens as hosts, and this infection can result with tumors in lymphoid [62] . Nowadays, it still is a serious threat for poultry industry, in addition to this, researches about the interaction between avian leucosis virus and immune system are really promising for the prevention and treatment of human retroviral diseases such as acquired immune deficiency syndrome (AIDS) [63] . HIV is the cause of AIDS which was first notified in 1981, and it is associated with Kaposi's sarcoma, non-Hodgkin's lymphoma, and invasive cervical cancer [62, 64] . With current vaccination and antiviral drug therapy, it is possible to protect from infection and prevent the progress of disease [62] . Human T-cell leukemia virus which is a RNA virus infects mainly T-cells and causes an endemic disease. It is also related with T-cell tumors [61] . Adenovirus: Adenovirus is an icosahedral, nonenveloped tumor virus with a double-stranded DNA [65, 66] . Adenovirus was first isolated from adenoid tissue of a human [65] . Human adenovirus with its subgroups of A, C, and E causes infections with mild symptoms such as respiratory infections, gastroenteritis, and conjunctivitis in humans [65, 66] . In addition to these, adenoviruses cause tumor in rodents but not in humans because when an adenovirus infects a human, the virus can complete its full life cycle in order to create a new virus [66] . But in rodents, such as mice, the virus cannot complete its life cycle at the late phase and that results with cell transformation [66] . Togavirus: Togaviruses are small, icosahedral, enveloped viruses with single-stranded RNA genome [57, 67] . Sindbis virus and Semliki Forest virus from Togaviruses which can infect birds and rodents are not serious pathogens for humans, but they are significant veterinary pathogens. Among all togaviruses, only Rubella virus causes an important infection in humans, which is German measles [57, 67] . Transmission of togaviruses occurs with mosquitoes [57, 67] . Chikungunya virus is a novel virus that uses primates and mosquitoes as hosts and causes fever, arthritis, and rash in humans [57] . Its geographical distribution involves Africa, India, and Southeast Asia [67] . Venezuelan equine encephalitis virus (VEEV) is a zoonotic virus whose hosts are birds, horses, mosquitoes, and humans [57, 67] . VEEV causes fever and encephalitis in humans, and its transmission cycle consists of mosquitoÀhorseÀmosquito and spreads throughout humans [57, 67] . Astrovirus: Astroviruses from Astroviridae family have nonenveloped, icosahedral, single-stranded RNA virions [68] . First observation of Astroviruses goes back to 1975; they were found in the feces of infants with diarrhea [68, 69] . After that, with the improvement in the identification methods for viruses such as immunoassays and PCR, they have been described as the most common viral gastroenteritis (community or hospital acquired) agent in children younger than 2 years of age after rotavirus and calicivirus [68À70]. Astroviruses are transmitted by the fecalÀoral route, and their infections are not common in adults [69] . Astroviruses-related gastroenteritis causes only mild symptoms, and it can be treated with hydration and electrolyte replacement without further medical treatment [70] . Calicivirus: Caliciviruses from family of Caliciviridae are small, icosahedral, nonenveloped, and single-stranded RNA viruses, which were first identified using immunoelectron microscopy in 1972 [71] . Nowadays, caliciviruses are considered the primal causing agent of gastroenteritis with astroviruses and rotaviruses [71] . They can affect both adults and younger children and cause severe childhood diarrhea [71] . Rotavirus: Rotaviruses are large, icosahedral, nonenveloped, single-stranded RNA viruses, and they are members of Reoviridae family [57] . First observation and identification of rotavirus was in young children with gastroenteritis in 1973 by electron microscopy [72] . They are the leading agent of gastroenteritis that especially occurs in winter in the children under 5 years of age worldwide [57] . Rotavirus infections cause vomiting and diarrhea which result with dehydration, if not treated carefully, it can prove fatal for children [57] . Currently, Rotarix and RotaTeq are available rotavirus vaccines; nonetheless, there are no therapeutic antiviral drugs against rotavirus infection [72] . Papillomavirus: Papillomaviruses are small, nonenveloped, icosahedral, double-stranded DNA viruses from Papillomaviridae family [73, 74] . Their first identification goes back to the 1930s [75] . They can affect a wide variety of creatures from humans to monkeys and rabbits [73] . In humans, they infect genital tract causing cervical and vaginal cancers, respiratory tract, eye, mouth, and skin causing skin warts and skin cancer [75] . Human papillomavirus (HPV) is a carcinogenic virus that can be mucosal or cutaneous [73] . Gardasil and Cervarix are available HPV vaccines for prophylaxis [73] . For the clinical diagnosis of HPV infections, Pap smear procedure is used, which examines HPVrelated changes and malignancies in cells. Routine screening is really important for early diagnosis and treatment [75] . Picornavirus: Picornavirus is a small, nonenveloped, icosahedral RNA virus, which belongs to Picornaviridae family [76] . They can affect both animals and humans, picornavirus infections may be related with gastrointestinal tract, respiratory tract, muscles, and neuronal tissues [77] . Foot and mouth disease virus infects livestock such as cows, goats, sheep, etc. and causes foot and mouth disease by affecting epithelial cells [77] . Encephalomyocarditis is a disease which is caused by encephalomyocarditis virus (EMCV). EMCV infects heart and central nervous system in humans [77] . Poliovirus is one of the first human viruses, the structure of which was examined by X-ray crystallography [76] . It is the causing agent of poliomyelitis in humans which is a paralytic disease resulting from the destruction of neurons in the spinal cord [77] . Rhinovirus infects upper and lower airway tract in humans and causes colds and respiratory diseases [76, 77] . Hepatitis A virus was first identified as the agent of hepatitis A in 1973 [76] . It infects parenchymal cells of liver in humans [76] . Orthomyxovirus: Orthomyxoviruses are spherical, enveloped, single-stranded RNA viruses. Influenza viruses A, B, C, and D are the most significant types of orthomyxoviruses, and they cause infections in a wide variety of creatures from humans to birds. While influenza B virus can only infect humans, influenza A virus can infect not only humans but also pigs, horses, whales, seals, etc. In order to make a further categorization, subtypes can be formed based on the hosts of viruses. RT-PCR is the current detection method for the identification of influenza viruses. Influenza virus related influenza symptoms are described as sudden onset of fever, headache, malaise, sore throat, myalgia, and nonproductive cough. However, as a common diagnostic mistake, all respiratory illnesses might be considered as flu. For sensitive and accurate diagnosis of influenza, RT-PCR is used. Influenza viruses affect epithelial cells in the upper and lower respiratory tract, and this infection is extremely contiguous due to quick spread potential by coughing and sneezing of infected people. There are two types of available therapeutic antiviral drugs: neuraminidase inhibitors (oseltamivir and zanamivir) and drugs related with the interference of the function of viral envelope (amantadine and rimantadine). There are also prophylactic live attenuated vaccines such as LAIV4 and FluMist [78, 79] . Coronavirus: Coronaviruses are large, enveloped, spherical, single-stranded RNA viruses. Majority of coronaviruses are the causing agents of acute/chronic, lethal, zoonotic diseases of respiratory or enteric tracts such as otitis media, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS) by infecting epithelial cells. Transmission of SARS and MERS occurs with close contact. RT-PCR is used for diagnostic analysis of coronaviruses [80À82] . Rhabdovirus: Rhabdoviruses are long, enveloped, single-stranded RNA viruses from Mononegavirales [83] . Rabies virus and Vesicular stomatitis virus (VSV) are most important rhabdoviruses [83, 84] . Even though VSV, which is the causing agent of vesicular stomatitis, a zoonotic disease, is not considered an important pathogen for humans, it has gained significance due to its newly discovered oncolytic activity and possible use for cancer therapy [84] . Rabies virus was first studied by Louis Pasteur in 1885 even before the detailed studies on viruses have started and he developed a postexposure vaccine [85] . Rabies virus is the only significant human pathogen of rhabdoviruses [83, 85] . It infects the central nervous system of humans and animals and causes fatal disease, rabies, leading to fatal encephalomyelitis [83] . At the onset of the rabies the symptoms are mild and flu-like, but in the later phases of disease, symptoms get more severe such as paralysis, anxiety, insomnia, and hydrophobia; at this stage the recovery is almost impossible [83] . There are currently available postexposure vaccines for humans, domestic, and wildlife animals [84] . Hepadnavirus: Hepadnaviruses are small, spherical, enveloped DNA viruses from Hepadnaviridae family [86] . HBV, as the smallest human DNA virus and the causing agent of acute viral hepatitis with chronic liver disease and hepatocellular carcinoma, is the most important hepadnavirus [86À88]. Transmission route of HBV is through body fluids, such as blood, saliva, and vaginal fluids [87] . Recombinant vaccines for hepatitis B prophylaxis is first injected after birth and then during childhood [87] . Vaccines provide high ratio of protection and immunity against HBV, and they decrease the transmission and prevalence of HBV worldwide [87] . Flavivirus: Flaviviruses are enveloped, icosahedral, single-stranded RNA viruses which include Dengue virus (DENV) and hepatitis C virus (HCV) [89, 90] . Important flaviviruses that cause infections in humans are listed next: mosquitoes are the transmission agent of yellow fever virus (YFV) to humans [90] . YFV causes hemorrhagic fever with accompanying symptoms, such as vomiting, back pain, and photophobia, and patients to look yellow [89À91]. DENV is classified into four types, and the transmission of all these to humans takes place through mosquitoes [90] . DENV infection results with hemorrhagic fever and dengue shock syndrome [90] . With early diagnosis and accurate treatment, fatality rate can be decreased [90] . Japanese encephalitis virus (JEV) is also transmitted to humans through mosquitoes, such as YFV and DENV, and it is the main cause of encephalitis in Asia [89, 90] . Vaccines against JEV are available in Asia and Australia [90] . HCV, which causes acute/chronic hepatitis and liver cancer resulting immunosuppression, was first discovered in 1989 [89] . Ribavirin, sofosbuvir, alpha interferon, and boceprevir are the currently used antiviral drugs for HCV; nonetheless, there is no available preventive vaccine [89] . Zika virus (ZIKV) was first isolated in 1947, but the first ZIKV outbreak took place in 2007. After that, by the end of 2016, ZIKV cases have started to spread rapidly and WHO declared Public Health Emergency of International Concern. General symptoms of ZIKV infections are vomiting, edema, myalgia, headache, and fever. Researches on the development of vaccines against ZIKV are still an ongoing process [90] . Biosensor-related research has received high attention over the last three decades. Biosensors have some advantages, such as affordable, fast responsive, and easy to operate analytical-friendly techniques. Therefore they present a wide area of detection and diagnosis suited for health-care analysis. Biosensors are commonly defined as analytical devices composed of a biological recognition system [92] and a physicochemical transducer [93] . Biosensors have highly selective properties due to possiblity of tailoring the specific interaction of compounds by immobilizing biological recognition elements on the sensor [94] . Typically biosensors comprises three components: a bioreceptor or biological identification component, a signal transducer, and an amplifier [94] (Fig. 18.1) . A biosensor is an analytical device, which converts biological response into a quantifiable and processable signal [96] . Immobilizing a biologically sensitive material on the surface of a biosensor is a new approach in biosensor technology. Bioreceptor elements are generally considered as biomarker, enzymes, microorganism, nucleic acids, tissues, virus, bacteria, and antigens [97] . The most common traditional techniques, such as electrochemical [cyclic voltammetry (CV), amperometric, impedance spectroscopy, potentiometric], optical, and various field-effect transistor-based methods, are described [96, 98] . Nanomaterials (NMs) bring new possibilities for the development of electrochemical biosensors. Incorporation of NMs with promising novel approaches in biosensor design provides construction of biosensors and development of novel electrochemical assays [99, 100] . In addition, the advanced nanoscale biosensor can be utilized to achieve high sensitivity and selectivity of biological sensing for analytical purposes in various fields of research and technology [92, 95] . NMs have great potential due to its promoting electron transfer reactions, high surface area and electrical conductivity, good chemical stability, and mechanical robustness [101] . Moreover, they can be used to enhance electrochemical reaction and promote signal of biorecognition system [100] (Figs. 18.2 and 18.3) . Various NMs, such as magnetic NPs (iron oxide NPs), metal NPs (gold and silver NPs), carbon-based nanotubes and carbon allotropes, nanowire, and quantum dots with different biological recognition elements (enzymes, nucleic acids, antibodies, antigens, peptide), provide many opportunities for enhancing the performance of nanobiosensor [104, 105] . The electrochemical nanobiosensors were used in versatile areas of cancer diagnostics and detection of infectious microorganisms, virus, etc. [100] . In sensing substrates, molecular recognition processes play a central role in biosensors. Substrates of biosensors enhance the performance of the biosensors. Moreover, the sensing substrates improve the biosensing sensitivity, specificity, stability, and response dynamics [106] . There are three main engineering techniques to construct highly efficient sensing substrates: nanostructured sensing substrates, molecule-mediated interface, and DNA nanostructureÀfunctionalized sensing interface [106] . Nanostructured sensing substrates are generally used for ultrasensitive detection of nucleic acids and proteins. Molecule-mediated interface is especially applied for small molecules. The biosensors have a wide range of analytes from small molecules to proteins, such as enzymes, antibodies, or oligonucleotides. Biological materials can be immobilized on nanostructured electrodes surface. For example, oligonucleotides are interacted with electron surface for designing biosensors genomic analysis [107] . Immunosensors are based on a working principle-the specific antigenÀantibody interaction connected with different transducers. Most of the immunosensors are based on a direct and indirect format and labeled methods. The specific antigens are first immobilized on the electrode surface, and then the analyte (antibody) is added on antigen linked electrode surface. Therefore the specific antigen selectively recognizes and binds the antibody, and the specific anti-genÀantibody complex can be evaluated using secondary antibody labeled with an enzyme [108] . Electrochemical design of nucleic acids biosensor is closely related to DNA sequencing methods based on genomics [109] . The application of nucleic acids as a bioreceptor manages analysis of RNA/DNA-based biosensors. Moreover, nucleic acid bioreceptors are also employed for detection of pathogens regards to complementary base pairs [110] . Biosensors are advantageous in relation to other PCR product-analysis techniques because they can add speed and precision to the molecular assay and can also perform simultaneous analysis of multiple analytes [111] . Biosensors can be mainly classified as optical, electrochemical, and mass spectrometric. Among biosensors, electrochemical biosensors with a volumetric transducer displayed a great potential in the detection of biomolecules [112] . Electrochemical biosensors are widely used for the detection of various analytes because of their rapid response, great sensitivity, simplicity, cost-effective, miniaturization, and portable. Fabrication of biosensors using the techniques of square wave voltammetry (SWV), CV, and electrochemical impedance spectroscopy (EIS) allows rapid biosensing for different types of analytes. EIS has enlighten at surface area and interaction modification agents and the electrode surface [113] . Because of occurrence of an electrochemical reaction at the electrode surface upon interaction with target molecule, impedance biosensors have been widely used for the environmental monitoring of disrupting chemicals and drugs, interaction between antibody and antigen, and DNA strains [114] . The EIS measurement studies the dielectric parameters of a biological system in wide frequencies. The EIS provides information about surface adsorption, ion exchange, diffusion, and charge transfer [114] . In another electrochemical technique, quantitative analysis using SWV is one of the most promising mechanisms in the fabrication of biosensors due to their ability to perform more sensitive answer for rapid biosensing when compared to differential pulse voltammetry (DPV) techniques. In pulse methods the procedures are based on the application of pulse changes of potential, and the current response is measured at a suitable time relative to the time of the pulse [115] . All pulse techniques are based on the difference in the rate of the decay of the charging and the faradaic currents related a potential step or pulse. The working electrode (WE) represents the fundamental component in electrochemical studies. The most commonly used WE materials are metal electrodes (Pt, Au, Hg, etc.) and carbon electrodes. It can be worked in more negative potential area by using carbon electrodes as well as good anodic potential windows. The most common form of carbon electrode is glassy carbon electrode. Moreover, carbon paste electrodes are also useful in many applications [116] . Siuzdak et al. worked on pathogen-detection methods. The nanocrystalline boron-doped diamond-based electrode (B:NCD) was used as platform of biosensor. The modified material is a highly promising material for the thirdgeneration biosensor due to its chemical inertness, wide potential window, low background current, biocompatibility, and high stability [117] . H5N1, Avian influenza virus, is determined by different methods such as the immunochromatography, the reversetranscription PCR (RT-PCR), ELISA, serological methods [9] , quartz crystal microbalance, surface plasmon resonance, and fluorescence. However, the electrochemical detection method received high attention because of providing low cost, small sample volume without amplification step, and user-friendly interface and portability [118] . Lee et al. reported the H5N1 detection with 1 pM of limit of detection (LOD). CV was applied to confirm the HA protein binding to multifunctional DNA structure on pAuNPs-modified electrode. Hepatitis B e antigen (HBeAg) immunosensor was developed using electrochemical techniques. Cocatalysis of horseradish peroxidase (HRP) and nanoporous gold are used as modifier agents. The developed immunosensor gives a good linear relation between peak current and concentration of HBeAg (1 pg/mL to 1 ng/mL as well as 0.064 pg/mL of LOD). The electrochemical DNA biosensing device becomes effective tool because of the properties such as rapid response time, high specificity, sensitivity, and user friendly. Electrochemical paper analytical device (ePADs) makes a great contribution on the sensor, because of paper being an inexpensive substrate. Singhal et al. describe their study about the fabrication of ePADs, by diagnosing the target DNA of Chikungunya virus (CHIKV) [119] . Electrical and optical methods were used in the study by Oliveira et al. [120] . Genomic DNA is detected in blood plasma of patients with hepatitis B without PCR amplification. The linear range of HBV-genomic DNA concentration was found as 1.55À6.68 ng/μL with LOD of 0.15 ng/μL. HBV surface antigen (HBsAg) immunosensor was developed by Alizadeh et al. Hemin/G-quadruplex/ Fe 3 O 4 ÀAuNPs and H-amino-rGOÀAu were used as modifier agents [121] . The HBsAg immunosensor was also applied in spiked human serum sample. Smart electrochemical platform was formed by Wiang et al. An ultrasensitive label-free electrochemical biosensor using graphene quantum dots for detecting HBV DNA was made. The proposed sensor exhibits high sensitivity with a detection limit of 1 nM, and the linear detection range is from 10 to 500 nM [122] . EIS detection is one of the main concepts in label-free biosensing which are nucleotides (DNA/RNA), enzymes, aptamers, and antibodies detection. Moreover, EIS measurement is a nondestructive and relatively facile system. Shariati et al. developed a biosensor of HPV DNA. Impedimetric HPV DNA biosensor by AuNTs-polycarbonate electrode was fabricated. Biosensor in label-free detection showed the good linear ranges of 0.01À1 mM [114] . Nanocelluloses could be used to immobilize antibodies, enzymes, and noble metal NPs, all of which could enhance electrochemical immunosensor performance. Surprisingly, very little work has been reported on the application of nanocelluloses in electrochemical immunoassays. Liu et al. aim to develop a sensitive novel sandwich-type electrochemical immunosensor for the measurement of Avian leukosis virus subgroup J [123] . In this chapter, we tried to collect and describe all reported biosensors for viruses by electrochemical methods. Generally, measurements depend on the affinity interaction between antibody and antigen. The most used methods were observed as amperometry, voltammetry, and impedance spectroscopy methods. The developed biosensors with viruses can be used with integrated substrates (metallic-or carbon-based electrodes) for clinical, environmental, and industrial applications. Some selected applications were summarized and listed in Table 18 .1. Handb. Water Wastewater Microbiol Modern Virology History of virology Structure and classification of viruses Viruses A label-free electrochemical immunosensor for hepatitis B based on hyaluronic acid-carbon nanotube hybrid film Label-free electrochemical immunosensor for direct detection of Citrus tristeza virus using modified gold electrode Early events in rabies virus infection-attachment, entry, and intracellular trafficking A Biological Perspective of Slow Virus Infection and Chronic Disease Laboratory diagnosis of virus diseases Methods to study viruses Multiplex polymerase chain reaction (PCR) and real-time multiplex PCR for the simultaneous detection of plant viruses Reverse transcription (RT) and polymerase chain reaction (PCR), Basic Mol Immunoblotting (Western blot), Basic Mol. Protoc. Neurosci. Tips Tricks Pitfalls Evaluation of immunoblotting method for the detection of antibodies to mumps virus Identification of poliovirus precursor proteins by immunoprecipitation Rapid purification of Soybean mosaic virus from small quantities of tissue by immunoprecipitation The Enzyme-Linked Immunosorbent Assay: The Application of ELISA in Clinical Research Immunoanalytical Technique: Enzyme-Linked Immunosorbent Assay (ELISA) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses Hemagglutination assay, in: Virology Detection and diagnosis of viral infections Comparison of enzyme-linked immunosorbent assay, hemagglutination inhibition, and passive latex agglutination for determination of rubella immune status Diagnosis and methods Flow virometry as a tool to study viruses Flow cytometry: surface markers and beyond Flow cytometric quantification of viruses in activated sludge Quantitative flow cytometry to measure viral production using infectious pancreatic necrosis virus as a model: a preliminary study Transmission electron microscopy, in: Bancroft's Theory Pract Virus quantitation by transmission electron microscopy, TCID50, and the role of timing virus harvesting: a case study of three animal viruses Viral detection by electron microscopy: past, present and future Recent studies on the fine structure of viruses by electron microscopy, using negative-staining techniques Comparison of direct and indirect immunofluorescence staining of clinical specimens for detection of respiratory syncytial virus antigen Plaque assays, Virology (2014) 74À84. Available from A simple and highly repeatable viral plaque assay for enterovirus 71 Development of an immunofluorescence focus assay for Ebola virus Supported protein G on gold electrode: characterization and immunosensor application Biosensors for hepatitis B virus detection A rapid-response ultrasensitive biosensor for influenza virus detection using antibody modified boron-doped diamond A DNA-based nanobiosensor for the rapid detection of the dengue virus in mosquito Electrochemical biosensors for influenza virus a detection: the potential of adaptation of these devices to POC systems Towards the electrochemical diagnostic of influenza virus: development of a graphene-Au hybrid nanocomposite modified influenza virus biosensor based on neuraminidase activity Electrochemical sensors and biosensors for influenza detection Human viruses: discovery and emergence Risk factors for human disease emergence Essent. Hum. Virol Mol. Virol. Hum. Pathog. Viruses Herpesviruses, Vascular Responses to Pathogens Aseptic and viral meningitis Measles virus; informations, Hum. Vaccine Immunother Other DNA viruses Natural pathogens of laboratory animals: their effects on research The pathogenesis of infection with minute virus of mice depends on expression of the small nonstructural protein NS2 and on the genotype of the allotropic determinants VP1 and VP2 Viruses Immunity to Avian leukosis virus: where are we now and what should we do? Front HIV (Human Immunodeficiency Virus) Adenoviruses: general features Mol. Virol. Hum. Pathog. Viruses Mucocutaneous Manifestations Viral Dis Mol. Virol. Hum. Pathog. Viruses Coronavirus pathogenesis, Adv. Virus Res The nonstructural proteins directing coronavirus RNA synthesis and processing Mol. Virol. Hum. Pathog. Viruses The impact of rapid evolution of hepatitis viruses Hepatitis viruses Mol. Virol. Hum. Pathog. Viruses Flaviviruses, Zika virus Advances in biosensors: principle, architecture and applications Recent trends in rapid detection of influenza infections by bio and nanobiosensor Nanobiosensors: point-of-care approaches for cancer diagnostics Electrochemical biosensors-sensor principles and architectures Helicobacter pylori point-of-care diagnosis: nano-scale biosensors and microfluidic systems Electrochemical nanobiosensors An electrochemical sensor based on silver nanoparticles-benzalkonium chloride for the voltammetric determination of antiviral drug tenofovir Cancer diagnosis using nanomaterials based electrochemical nanobiosensors Recent trends in nanomaterial-modified electrodes for electroanalytical applications Nano-biosensing approaches on tuberculosis: defy of aptamers Magnetic nanoparticle decorated graphene based electrochemical nanobiosensor for H 2 O 2 sensing using HRP Engineering electrochemical interface for biomolecular sensing Biosensor applications of electrodeposited nanostructures Electrochemical biosensors as potential diagnostic devices for autoimmune diseases Electrochemistry of nucleic acids Recent advances on application of peptide nucleic acids as a bioreceptor in biosensors development Disposable biosensors for clinical diagnosis Early detection of lung cancer biomarkers through biosensor technology: a review Impedance sensing of DNA immobilization and hybridization by microfabricated alumina nanopore membranes An ultrasensitive label free human papilloma virus DNA biosensor using gold nanotubes based on nanoporous polycarbonate in electrical alignment Electroanalytical methods for the determination of pharmaceuticals: a review of recent trends and developments Electrodes-Chemistry LibreTexts Biomolecular influenza virus detection based on the electrochemical impedance spectroscopy using the nanocrystalline boron-doped diamond electrodes with covalently bound antibodies Fabrication of electrochemical biosensor consisted of multifunctional DNA structure/porous au nanoparticle for avian influenza virus (H5N1) in chicken serum Paper based DNA biosensor for detection of chikungunya virus using gold shells coated magnetic nanocubes Application of nanomaterials for the electrical and optical detection of the hepatitis B virus Dual amplified electrochemical immunosensor for hepatitis B virus surface antigen detection using hemin/ G-quadruplex immobilized onto Fe 3 O 4 -AuNPs or (hemin-amino-rGO-Au) nanohybrid A label-free electrochemical platform for the highly sensitive detection of hepatitis B virus DNA using graphene quantum dots Electrochemical immunosensor with nanocellulose-Au composite assisted multiple signal amplification for detection of Avian leukosis virus subgroup Electrochemical immunosensor for HBe antigen detection based on a signal amplification strategy: the co-catalysis of horseradish peroxidase and nanoporous gold A colorimetric and electrochemical immunosensor for point-of-care detection of enterovirus 71 Electrochemical detection of plant virus using gold nanoparticlemodified electrodes Portable bioactive paper based genosensor incorporated with Zn-Ag nanoblooms for herpes detection at the point-of-care A photoelectrochemical immunosensor based on gold nanoparticles/ZnAgInS quaternary quantum dots for the high-performance determination of hepatitis B virus surface antigen Electrochemical detection of influenza virus H9N2 based on both immunomagnetic extraction and gold catalysis using an immobilization-free screen printed carbon microelectrode Gold nanoparticle-decorated reduced-graphene oxide targeting anti hepatitis B virus core antigen Ultrasensitive electrochemical immunosensor for quantitative detection of HBeAg using Au@Pd/MoS 2 @MWCNTs nanocomposite as enzyme-mimetic labels Evaluation of carbon nanotube based copper nanoparticle composite for the efficient detection of agroviruses An electrochemical immunosensor with graphene-oxide-ferrocene-based nanocomposites for hepatitis B surface antigen detection Biosensor-based selective detection of Zika virus specific antibodies in infected individuals Impedimetric quantification of anti-dengue antibodies using functional carbon nanotube deposits validated with blood plasma assays Magnetic bead/capture DNA/glucose-loaded nanoliposomes for amplifying the glucometer signal in the rapid screening of hepatitis C virus RNA Using boehmite nanoparticles as an undercoat, and riboflavin as a redox probe for immunosensor designing: ultrasensitive detection of hepatitis C virus core antigen Detection of Odontoglossum ringspot virus infected Phalaenopsis using a nano-structured biosensor A nanohybrid probe based on double recognition of an aptamer MIP grafted onto a MWCNTs-Chit nanocomposite for sensing hepatitis C virus core antigen Nucleic aptamer modified porous reduced graphene oxide/ MoS 2 based electrodes for viral detection: application to human papillomavirus (HPV) Langmuir-Blodgett nanoassemblies of the MoS 2 -Au composite at the air-water interface for dengue detection Hydroxyapatite nanoparticles modified graphite electrodes for electrochemical DNA detection Digital single virus electrochemical enzyme-linked immunoassay for ultrasensitive H7N9 avian influenza virus counting Facile synthesis of MoS 2 @Cu 2 O-Pt nanohybrid as enzyme-mimetic label for the detection of the hepatitis B surface antigen An electrochemical biosensor based on surface imprinting for Zika virus detection in serum Approaching single DNA molecule detection with an ultrasensitive electrochemical genosensor based on gold nanoparticles and cobalt-porphyrin DNA conjugates A glassy carbon immunoelectrode modified with vanadium oxide nanobelts for ultrasensitive voltammetric determination of the core antigen of hepatitis C virus Electrochemical biosensing of influenza A subtype genome based on meso/macroporous cobalt(II) oxide nanoflakes-applied to human samples A nano-Au/C-MWCNTbased label free amperometric immunosensor for the detection of capsicum chlorosis virus in bell pepper A high sensitive electrochemical avian influenza virus H7 biosensor based on CNTs/MoSx aerogel TiO 2 nanoparticles doped with Celestine Blue as a label in a sandwich immunoassay for the hepatitis C virus core antigen using a screen printed electrode Impedimetic biosensor for the DNA of the human papilloma virus based on the use of gold nanosheets An elegant analysis of white spot syndrome virus using a graphene oxide/methylene blue based electrochemical immunosensor platform A multi-walled carbon nanotubes based molecularly imprinted polymers electrochemical sensor for the sensitive determination of HIV-p24 Carbon nanoparticles based electrochemical biosensor strip for detection of Japanese encephalitis virus Label-free detection of influenza viruses using a reduced graphene oxide-based electrochemical immunosensor integrated with a microfluidic platform A novel method for dengue virus detection and antibody screening using a graphene-polymer based electrochemical biosensor Impedimetric genosensor for detection of hepatitis C virus (HCV1) DNA using viral probe on methylene blue doped silica nanoparticles Electrospun manganese(III) oxide nanofiber based electrochemical DNA-nanobiosensor for zeptomolar detection of dengue consensus primer Using silver nanoparticle and thiol graphene quantum dots nanocomposite as a substratum to load antibody for detection of hepatitis C virus core antigen: electrochemical oxidation of riboflavin was used as redox probe A highly sensitive electrochemical immunosensor for hepatitis B virus surface antigen detection based on hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme-signal amplification A genosensor for detection of consensus DNA sequence of Dengue virus using ZnO/Pt-Pd nanocomposites A needle-like Cu 2 CdSnS 4 alloy nanostructure-based integrated electrochemical biosensor for detecting the DNA of Dengue serotype 2 Ultra-sensitive aptasensor based on a GQD nanocomposite for detection of hepatitis C virus core antigen Label-free amperometric immunosensor based on graphene oxide and ferrocene-chitosan nanocomposites for detection of hepatitis B virus antigen Graphene oxide-polycarbonate track-etched nanosieve platform for sensitive detection of human immunodeficiency virus envelope glycoprotein Silver nanoparticles coated graphene electrochemical sensor for the ultrasensitive analysis of avian influenza virus H7 A multi-virus detectable microfluidic electrochemical immunosensor for simultaneous detection of H1N1, H5N1, and H7N9 virus using ZnO nanorods for sensitivity enhancement Chitosan-carbon nanofiber modified single-use graphite electrodes developed for electrochemical detection of DNA hybridization related to hepatitis B virus Ei, sensitive impedimetric immunoassay of Japanese encephalitis virus based on enzyme biocatalyzed precipitation on a gold nanoparticle-modified screen-printed carbon electrode Label-free electrochemical impedance detection of rotavirus based on immobilized antibodies on gold sononanoparticles Impedimetric genosensor for ultratrace detection of hepatitis B virus DNA in patient samples assisted by zeolites and MWCNT nano-composites Polymerase chain reaction-free detection of hepatitis B virus DNA using a nanostructured impedance biosensor Rapid detection of avian influenza virus H5N1 in chicken tracheal samples using an impedance aptasensor with gold nanoparticles for signal amplification Synergistic effect of magnetite and gold nanoparticles onto the response of a label-free impedimetric hepatitis B virus DNA biosensor Dual immunosensor based on methylene blue-electroadsorbed graphene oxide for rapid detection of the influenza A virus antigen A nanohybrid of platinum nanoparticles-porous ZnOÀhemin with electrocatalytic activity to construct an amplified immunosensor for detection of influenza Chronoamperometry measurement for rapid cucumber mosaic virus detection in plants A proof of concept: detection of avian influenza H5 gene by a graphene-enhanced electrochemical genosensor A regenerating ultrasensitive electrochemical impedance immunosensor for the detection of adenovirus Bifunctional magnetic nanobeads for sensitive detection of avian influenza A (H7N9) virus based on immunomagnetic separation and enzyme-induced metallization Simple in situ functionalizing of magnetite nanoparticles by 4-nitrobenzenediazonium for construction of a sensitive electrochemical DNA biosensor for detection of a DNA sequence related to Hepatitis B virus A sensitive electrochemical biosensor for specific DNA sequence detection based on flower-like VS2, graphene and Au nanoparticles signal amplification Ultrasensitive electrochemical biosensor for HIV gene detection based on graphene stabilized gold nanoclusters with exonuclease amplification Biosensor based on SiNWs/AuNPs modified-screen printed electrode for dengue virus DNA oligomer detection Electrochemical detection of dengue virus NS1 protein with a poly(allylamine)/carbon nanotube layered immunoelectrode Electrochemical DNA biosensor based on a tetrahedral nanostructure probe for the detection of avian influenza A (H7N9) virus Electrochemical DNA biosensor based on gold nanorods for detecting hepatitis B virus Amperometric bioaffinity sensing platform for avian influenza virus proteins with aptamer modified gold nanoparticles on carbon chips Reactive carbon nano-onion modified glassy carbon surfaces as DNA sensors for human papillomavirus oncogene detection with enhanced sensitivity Exploiting enzyme catalysis in ultra-low ion strength media for impedance biosensing of avian influenza virus using a bare interdigitated electrode A multiple-promoted silver enhancement strategy in electrochemical detection of target virus Biosensor based on nanocomposite material for pathogenic virus detection Functional magnetic nanoparticles for clinical application: electrochemical immunoassay of hepatitis B surface antigen and α-fetoprotein Electrochemical-DNA biosensor development based on a modified carbon electrode with gold nanoparticles for influenza a (H1N1) detection: effect of spacer Ultrasensitive electrochemical immunoassay for avian influenza subtype H5 using nanocomposite A highly sensitive and selective hepatitis B DNA biosensor using gold nanoparticle electrodeposition on an Au electrode and mercaptobenzaldehyde 3D printed chip for electrochemical detection of influenza virus labeled with CdS quantum dots Electrochemical immunosensor with graphene quantum dots and apoferritin-encapsulated Cu nanoparticles double-assisted signal amplification for detection of Avian leukosis virus subgroup Magnetic nanoparticle-based immunosensor for electrochemical detection of hepatitis B surface antigen Dengue virus detection using impedance measured across nanoporous alumina membrane Electrochemical detection of hepatitis B and papilloma virus DNAs using SWCNT array coated with gold nanoparticles Electrochemical sensor based on direct electron transfer of HIV-1 virus at Au nanoparticle modified ITO electrode Impedimetric DNA biosensor based on a nanoporous alumina membrane for the detection of the specific oligonucleotide sequence of dengue virus Sandwich immunoassay for hepatitis C virus non-structural 5A protein using a glassy carbon electrode modified with an Au-MoO 3 /chitosan nanocomposite Polyaniline nanowires-based electrochemical immunosensor for label free detection of Japanese encephalitis virus An ultrasensitive electrochemical immunosensor for HIV p24 based on Fe 3 O 4 @SiO 2 nanomagnetic probes and nanogold colloid-labeled enzymeÀantibody copolymer as signal tag Ultrasensitive norovirus detection using DNA aptasensor technology MultisHRP-DNA-coated CMWNTs as signal labels for an ultrasensitive hepatitis C virus core antigen electrochemical immunosensor A sensor tip based on carbon nanotube-ink printed electrode for the dengue virus NS1 protein Electrochemical DNA biosensor improved by imidazo[4,5-f]1,10-phenanthroline iron(iii) as an indicator based on pt-nanoparticles and carbon nanotubes modified electrode Multifunctional Fe 3 O 4 core/Ni-Al layered double hydroxides shell nanospheres as labels for ultrasensitive electrochemical immunoassay of subgroup J of Avian leukosis virus Development of an electrochemical membrane-based nanobiosensor for ultrasensitive detection of dengue virus Electrochemical impedance spectroscopy characterization of nanoporous alumina dengue virus biosensor Label-free sandwich type of immunosensor for hepatitis C virus core antigen based on the use of gold nanoparticles on a nanostructured metal oxide surface Ultrasensitive cDNA detection of dengue virus RNA using electrochemical nanoporous membrane-based biosensor Electrochemical detection of short HIV sequences on chitosan/Fe 3 O 4 nanoparticle based screen printed electrodes Development of interdigitated arrays coated with functional polyaniline/ MWCNT for electrochemical biodetection: application for human papilloma virus Impedimetric immunosensor for detection of plum pox virus in plant extracts Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode