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         MDPI

Diagnosis of Dengue Infection Using Conventional and Biosensor Based Techniques

         10.3390/v7102877

contrib:
            	  CONT

            Om Parkash

            Rafidah Hanim Shueb

            	  CONT

                  Ploss
                  Alexander,

Academic Editor

         [om.aprembhagat@gmail.com]
         []
         []
         [2015-10-19]
         [Copyright 2015: ]
         [Licence This article is an open access article distributed under the terms and conditions
            of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).]

         []
         []
         []

Abstract

Dengue is an arthropod-borne viral disease caused by four antigenically different
            serotypes of dengue virus. This disease is considered as a major public health concern
            around the world. Currently, there is no licensed vaccine or antiviral drug available
            for the prevention and treatment of dengue disease. Moreover, clinical features of
            dengue are indistinguishable from other infectious diseases such as malaria, chikungunya,
            rickettsia and leptospira. Therefore, prompt and accurate laboratory diagnostic test
            is urgently required for disease confirmation and patient triage. The traditional
            diagnostic techniques for the dengue virus are viral detection in cell culture, serological
            testing, and RNA amplification using reverse transcriptase PCR. This paper discusses
            the conventional laboratory methods used for the diagnosis of dengue during the acute
            and convalescent phase and highlights the advantages and limitations of these routine
            laboratory tests. Subsequently, the biosensor based assays developed using various
            transducers for the detection of dengue are also reviewed.

1. Introduction

Dengue, a vector-borne disease, is prevalent in tropical and sub-tropical regions
            of the world and associated with endemic as well as in epidemic transmission cycles.
            Dengue usually causes inapparent infection, but it could also lead to other wide range
            of clinical symptoms/diseases including mild dengue fever and severe dengue [

                  1

            ]. This disease is caused by dengue virus (DENV), which is a member of the
            Flavivirus genus within the
            Flaviviridae family. This virus is classified into four antigenically related but genetically
            distinctive serotypes, DENV-1, -2, -3 and DENV-4. The four DENV serotypes differ in
            the nucleotide sequence by 25–35 base pairs and each serotype is capable of causing
            dengue. Out of these four different serotypes, DENV-4 appears to be the most divergent
            serotype followed by DENV-2 while DENV-1 and DENV-3 are more closely related [

                  2

            ]. Infection with any serotype provides long-term immunity to that specific serotype
            only, but limited and temporary immunity to the other three serotypes [

                  3

            ,

                  4

            ]. Epidemiological studies have shown that secondary infection with different serotypes
            may lead to more severe dengue [

                  5

            ].

DENV is surrounded by an envelope which encloses single-stranded positive sense RNA
            comprising approximately 1100 nucleotide base. Translation of viral RNA produces a
            single polypeptide, which upon the proteolytic cleavage by proteases results in the
            formation of three structural proteins (capsid, membrane and envelope) and seven non-structural
            (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5) [

                  6

            ,

                  7

            ]. The structural proteins form the coat of the virus and help in delivering the RNA
            to target host cell. The non-structural proteins organize the production of a new
            virus in the host cell [

                  8

            ]. Dengue is spread between people by the mosquitoes
            Aedes aegypti and
            Aedes albopictus, through a human-to-mosquito-to-human cycle of transmission [

                  9

            ]. DENV enters into the blood through a mosquito bite and infects the peripheral blood
            mononuclear cells. Clinical symptoms typically appear 4–7 days following mosquito
            bite and may persist for 3–10 days [

                  10

            ].

2. Dengue as a Global Health Concern

Dengue is a serious health issue in tropical and subtropical countries around the
            world. Currently, the disease is endemic in more than 100 countries and imperils more
            than half of the world population. Three hundred and ninety million dengue infections
            are estimated to occur every year, and of that, 96 million are serious enough for
            patients to seek medical attention [

                  1

            ,

                  11

            ,

                  12

            ]. The dengue case burden and the number of countries reporting outbreaks have increased
            10-fold in the last 30 years [

                  13

            ]. This disease is now endemic in Africa, Americas, the Eastern Mediterranean, Southeast
            Asia and the Western Pacific. Southeast Asia and the Western Pacific are the most
            seriously affected areas with the former region contributes 52% cases annually [

                  6

            ,

                  14

            ,

                  15

            ]. Consequently, dengue inflicts a significant health, economic and social burden
            on the populations of endemic areas. A recent survey conducted in 12 countries within
            the South East Asia region revealed an annual economic burden of nearly 1 billion
            dollar incurred due to dengue [

                  16

            ].

3. Causes for Emergence of Dengue

There are several factors associated with the current increase of dengue incidence
            including uncontrolled growth of urban population, rapid urbanization, lack of vector
            control in dengue endemic areas, increased air travel, and inadequate public health
            care systems such as sewage and waste management [

                  17

            ,

                  18

            ]. Other important factors include temperature, humidity and rainfall [

                  19

            ].

4. A Brief History of Dengue

The word "dengue" comes from the Swahili language, which means a disease caused by
            an evil spirit. Although dengue-like disease was reported from China as far back as
            992 A.D, the first major dengue epidemics were only reported in the late 17th century
            [

                  20

            ]. The occurrence of dengue as an epidemic was not frequent at that time. Nonetheless,
            high incidence of severe dengue was demonstrated in the South-East Asia region after
            World War II, as urbanization provided a suitable condition for virus propagation
            [

                  21

            ]. In the South-East Asia, outbreaks of dengue fever were initially reported in 1954
            and 1956 in Manila, Philippines, and in 1958 in Bangkok [

                  22

            ]. The first epidemic in Singapore was recorded in 1960 with a low mortality rate
            [

                  23

            ]. During the 1960s and 1970s, the disease caused outbreaks in India, Malaysia, Singapore,
            Vietnam, Indonesia and Myanmar. At present, dengue has spread west into Pakistan,
            Sri Lanka, Maldives and east into Taiwan and China [

                  22

            ].

5. Laboratory Diagnosis and Its Significance

Clinical features of dengue infection, during acute and convalescent, are quite non-specific
            and mimic many other diseases (

               Table 1
            ). Thus, could be easily misinterpreted. Moreover, there is no specific treatment
            available for this disease. Thus, accurate laboratory diagnosis is very helpful in
            controlling this disease [

                  12

            ]. An early diagnosis helps in prompt patient management and immediate implementation
            of appropriate vector control measures which in turn helps to prevent the spread of
            the infection. Additionally, diagnostics provide key data on the epidemiology and
            health burden of dengue, which is very useful for accurate public health surveillance
            [

                  24

            ]. Currently, many in-house assays or rapid diagnostic tests (RDTs) have been developed
            are commercially available and used in routine diagnostic laboratories. However, selection
            of a proper test is dependent on various factors such as viremia period and infection
            status (primary or secondary) (
            Figure 1).

viruses-07-02877-t001_Table 1

            Table 1

Differential diagnosis for dengue illness.

                     Clinical symptoms
                     Differential diagnoses

                     Flu-like
                     Influenza, Measles, Chikungunya, Adenovirus infection, Infectious mononucleosis, Acute
                        HIV seroconversion illness

                     Rash
                     Rubella, Zika fever, West Nile fever, Measles, Scarlet fever, Meningococcal infection,
                        Chikungunya, Drug

                     Diarrhoea
                     Rotavirus infection, Norovirus, Cytomegalovirus, Viral hepatitis, Food poisoning

                     Muscle/Joint pain
                     West Nile fever, Zika fever, Chikungunya

                     Neurological manifestation
                     Meningoencephalitis, Tick-borne encephalitis, West Nile fever, Japanese encephalitis,
                        Febrile seizures

                     Bleeding tendency
                     Yellow fever, Ebola, Marburg

                     Thrombocytopenia
                     Rubella, Epstein-Barr virus, Parvovirus

            Figure 1

Suitability of dengue diagnostics at different phases of illness.

5.1. Conventional Diagnosis Methods

Several conventional laboratory techniques/tools are available for the diagnosis of
               dengue. Confirmation of dengue could be made through virus isolation, genome amplification
               as well as antigen and antibody detection via serology.

5.1.1. Viral Culture

Virus isolation has always been considered as the gold standard for the diagnosis
                  of dengue infection [

                        25

                  ]. For DENV cultivation, three methods have been reported, including inoculation of
                  specimens into mosquitoes, various
                  in vitro cultured cell lines, and intracerebrally in mice. Usually, common specimens including
                  plasma, serum, peripheral blood, cerebrospinal fluid, pleural fluid and immune system
                  tissues such as the liver, spleen and lymph node are used for virus culture. After
                  the cultivation of the specimen, a confirmation assay that includes immunofluorescence
                  assay or reverse transcriptase polymerase chain reaction (RT-PCR) is performed once
                  cytopathic effect in infected cells is observed. Although virus isolation method provides
                  high specificity, various practical aspects limit its use. First, this technique is
                  tedious and requires long incubation time (7–12 days) for virus cultivation and confirmation
                  [

                        26

                  ]. Second, it requires appropriate lab facilities with well-trained personnel. Third,
                  low level of virus titre in serum or blood is not suitable for virus culture [

                        11

                  ]. Lastly, the optimal window for culturing the virus is limited to 0–7 days following
                  the onset of symptoms, as DENV is detectable only during the acute phase of infection,
                  prior to development of dengue specific antibody response [

                        27

                  ,

                        28

                  ]. Therefore, despite being the gold standard for identification of dengue infection,
                  this approach is not practical in routine diagnostic laboratories.

5.1.2. Nucleic Acid Amplification

Detection of DENV by nucleic acid amplification using RT-PCR is suggestive of an acute
                  infection [

                        29

                  ]. This technique provides several advantages including ability to differentiate DENV
                  serotypes, can be a quantitative assay and has higher sensitivity when combined with
                  real-time technology [

                        29

                  ]. However, the RT-PCR test is expensive and requires specialized equipment and well-trained
                  personnel, thus, limiting its use in many developing countries [

                        30

                  ,

                        31

                  ,

                        32

                  ,

                        33

                  ].

5.1.3. Serological Diagnosis

Commercial serological assays are commonly used in diagnostic laboratories for dengue
                  confirmations. Serological assays are comparatively simple to perform and the specimens
                  required for the assay, such as serum or plasma, are stable in the tropical climate.
                  Consequently, these techniques can be used in various settings such as surveillance,
                  health care facilities and travel clinics. However, the applicability of serological
                  tests in dengue endemic areas should be evaluated against the potential cross-reactivity
                  with other circulating flaviviruses [

                        34

                  ].

Serological tests are more widely used for the detection of dengue infections in resources
                  limited countries as they are relatively inexpensive and easy to handle. These tests
                  include hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay
                  (ELISA) to detect immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies. RDTs
                  for some of these tests are also available commercially.

HI Assay

This test used to be very popular and acted as the gold standard for the diagnosis
                     of dengue. The principle of this test is based on the ability of dengue envelope protein
                     to agglutinate red blood cells. Anti-dengue antibodies present in dengue-infected
                     patient sera inhibit this agglutination and the extent of this inhibition is measured
                     in HI test [

                           35

                     ]. HI antibodies last for long periods, and thus, this test is very useful for seroepidemiologic
                     studies [

                           36

                     ]. The HI test can be used to detect and differentiate between primary (gradual increase
                     to moderate titer of antibody) and secondary (rapid increase to high titer) DENV infections
                     due to its simplicity and sensitivity [

                           28

                     ,

                           37

                     ]. HI antibody titers during primary infections peak at 1:640 whereas titers of 1:1280
                     or greater are common during secondary infections [

                           38

                     ]. The test was commercially developed into a simple, rapid, sensitive and specific
                     serological kit. However, the test was eventually replaced by ELISA based method for
                     the detection of dengue specific IgM and IgG antibodies due to its numerous practical
                     limitations [

                           39

                     ,

                           40

                     ]. Firstly, it cannot provide prompt diagnosis since paired sera are required. Secondly,
                     HI test also exhibits high cross-reactivity, resulting in impractical application
                     in countries where flavivirus infections are endemic. Lastly, it requires chemical
                     pre-treatment to remove nonspecific inhibitors of haemagglutination [

                           36

                     ,

                           37

                     ,

                           41

                     ,

                           42

                     ].

Detection of Immunoglobulin M (IgM) Antibody

The detection of dengue-specific IgM antibody is a useful diagnostic tool in resource
                     limited countries, particularly after a very short viremia period [

                           38

                     ]. During primary DENV infection, IgM antibody is the first immunoglobulin isotype
                     to appear while IgG antibody appears after a few days of IgM appearance. In contrast,
                     during secondary DENV infection, IgG antibody is the first to appear after onset of
                     symptoms while IgM antibodies appear after a few days at low titer or even undetectable
                     in some patients [

                           28

                     ]. Therefore, the presence of IgG and low levels of IgM during secondary infection
                     often obstruct accurate diagnosis when direct IgM ELISA is used. Consequently, dengue
                     IgM capture assays (MAC-ELISA) were designed to overcome the antigen-binding competition
                     between IgG and IgM and MAC-ELISA has now become a widely used method for the detection
                     of anti-DENV IgM antibodies [

                           43

                     ]. This method shows a sensitivity and specificity of 90% and 98%, respectively, in
                     samples collected after seroconversion [

                           44

                     ,

                           45

                     ].

RDTs based on lateral flow and particle agglutination format that provides result
                     as early as 15 min have also been developed. However, the sensitivity range of RDTs
                     in comparison to ELISA suggest that it is less sensitive test (

                        Table 2
                     ). Although due to the advent of technology, newer generation of RDTs and ELISA may
                     have higher sensitivity, the major drawbacks of all IgM antibody-based assays are
                     that they can cross-react with other flaviviruses and they may provide inaccurate
                     results if patients had recent dengue infection.

viruses-07-02877-t002_Table 2

                     Table 2

Sensitivity and specificity of evaluated commercialized dengue specific IgM ELISA
                           and rapid test.

                              Test format
                              Brand
                              Assay time
                              Sensitivity
                              Specificity
                              Ref.

                              ELISA
                              Dengue Fever Virus IgM Capture, Focus Diagnostics
                              225
                              98.6
                              79.9
                              [

                                       46

                                 ]

                              Pathozyme M Dengue, Omega
                              120
                              61.5
                              84.6
                              [

                                       46

                                 ]

                              Pathozyme M Dengue Capture, Omega
                              110
                              83.5
                              86.5
                              [

                                       45

                                 ]

                              Dengue IgM Capture, Panbio
                              130
                              89.5

87.6

                              861

88.1

                              [

                                       45

                                 ,

                                       47

                                 ]

                              SD Dengue IgM Capture, Standard Diagnostics
                              130
                              84.9
                              97.3
                              [

                                       47

                                 ]

                              InBios IgM ELISA
                              90
                              88.7
                              93.1
                              [

                                       48

                                 ]

                              Rapid test
                              Panbio Dengue Duo Cassette (IgM/IgG)
                              15
                              77.8

92.1

                              90.6

62.2

                              [

                                       46

                                 ,

                                       49

                                 ]

                              Hapalyse dengue-M PA kit, Pentax
                              90
                              97.7
                              76.6
                              [

                                       46

                                 ]

                              SD Bioline Dengue IgG/IgM
                              15–20
                              60.9

87.3%

                              90

86.8

                              [

                                       46

                                 ,

                                       49

                                 ]

                              Dengue check WB, Zephyr (IgM/IgG)
                              15
                              20.5
                              86.7
                              [

                                       46

                                 ]

Detection of IgG Antibody

Dengue specific IgG ELISA is introduced as an alternative to HI assay. The dengue
                     IgG capture ELISA (GAC-ELISA) is the most commonly employed format, in addition to
                     indirect IgG ELISA detection method. The IgG ELISA assay uses similar polyvalent viral
                     antigen as the MAC-ELISA and has a good correlation with HI assay. Meanwhile, IgG
                     avidity test can be used to classify between primary and a secondary dengue infection
                     in patient sera. This avidity test is based on the principle that the first antibodies
                     produced after primary infection exhibit low avidity (binding affinity) to an antigen
                     than those produced later [

                           38

                     ,

                           50

                     ,

                           51

                     ]. The IgG ELISA offers several advantages such as rapid, easy to perform and is suitable
                     for large-scale surveillance studies [

                           52

                     ]. Moreover, IgG based assay shows higher sensitivity than HI assay. However, similar
                     to the HI test, the IgG-based ELISA assay displays the same broad cross-reactivity
                     with other circulating flaviviruses. Additionally, this serological method could not
                     identify the infecting DENV serotype [

                           38

                     ].

Detection of DENV NS1 Antigen

DENV NS1 is a highly conserved glycoprotein, expressed as both membrane-associated
                     and secreted forms. Secreted NS1 has been detected ranging from 2–0.04 µg·mL
                     −1 in the serum of dengue-infected patients during the early stages of the disease [

                           53

                     ,

                           54

                     ,

                           55

                     ]. A high NS1 level has been demonstrated to circulate as early as one day after onset
                     of symptoms up to early convalescences, thus provides an alternative to virus culture
                     or PCR for early dengue diagnosis when IgM or IgG antibodies are not present yet in
                     dengue infected patients [

                           56

                     ].

Circulating dengue NS1 in sera can be detected either using ELISA assay or lateral
                     flow based RDTs. Several kits manufactured by various companies are available in the
                     market although sensitivity and specificity vary between them. A number of studies
                     have evaluated the sensitivity and specificity of these commercially available dengue
                     NS1 ELISA and RDTs (

                        Table 3
                     ) [

                           56

                     ].

viruses-07-02877-t003_Table 3

                     Table 3

Sensitivity and specificity of evaluated commercialized NS1 ELISA and rapid test.

                              Test format
                              Brand
                              Assay time (Min)
                              Sensitivity %
                              Specificity %
                              Ref.

                              ELISA
                              Platelia Dengue NS1 Ag Kit, Biorad
                              140
                              89.4

83.6

                              97.4

98.7

                              [

                                       26

                                 ,

                                       57

                                 ]

                              DENV Detect NS1 ELISA, Inbios
                              111
                              95.9
                              100
                              [

                                       57

                                 ]

                              Pan-E Dengue Early ELISA, Panbio
                              160
                              85.5

72.3

                              95.5

100

                              [

                                       26

                                 ,

                                       57

                                 ]

                              SD Dengue NS1 Ag ELISA
                              160
                              76.7
                              98.31
                              [

                                       58

                                 ]

                              Rapid test
                              Dengue NS1 Detect , Inbios
                              30
                              86.0

76.5

                              100

97.4

                              [

                                       57

                                 ,

                                       59

                                 ]

                              Biorad NS1 Ag Strip
                              15–30
                              72.8

79.1

                              100

100

                              [

                                       57

                                 ,

                                       59

                                 ]

                              Panbio NS1 Ag Strip
                              15
                              71.9
                              95
                              [

                                       57

                                 ]

                              SD Dengue Duo
                              15–20
                              70.6

72.4

                              100

73.4

                              [

                                       26

                                 ,

                                       57

                                 ]

5.2. Biosensor Based Methods for Dengue

Detection of IgM and NS1 based on RDTs and ELISA methods are the most widely used
               dengue assays in many countries. Although ELISA based assay have been shown to be
               more sensitive than RDTs, the former test lacks portability and thus, preventing its
               use in private clinics and field work. Because of this, many researchers have delved
               into biosensors as alternative new technology for the detection of DENV and dengue
               antibodies since this technique has several advantages such as higher sensitivity,
               cost-effective, simple fabrication, possible miniaturization, rapid outcome with quantitative
               analysis and possible on-site monitoring [

                     60

               ]. The most well-known commercially available biosensors are the glucose sensor and
               home pregnancy test [

                     61

               ]. However, unlike RDTs and ELISA based assay, development of biosensor for dengue
               diagnosis is still in its infancy stage and has not been commercialised. Based on
               different types of transducer, this section discusses three main types of biosensor,
               electrochemical, piezoelectric and optical, which have been developed for the diagnosis
               of dengue infection.

5.2.1. Electrochemical Biosensor

Electrochemical biosensors monitor the measurable current signals generated following
                  electrochemical oxidation and reduction reactions. The current signals recorded during
                  the reaction resulting from the recognition of a target analyte and measurement of
                  signals is proportional to target analyte concentrations [

                        62

                  ]. Among the reported electrochemical biosensor was lectin based electrochemical impedance
                  spectroscopy analysis developed to discriminate serum glycoproteins in dengue-infected
                  patients. The lectin was introduced as an alternative to a monoclonal antibody to
                  detect dengue serum glycoprotein (SG). Lectins are carbohydrate-binding proteins that
                  are highly specific for carbohydrate structure. In one study, concanavalin A (Con
                  A) lectin, a type of lectin specific to Gly/Man lectin and can be used for the recognition
                  of serum glycoprotein, was employed. This lectin was immobilized on the gold electrodes
                  through gold nanoparticles (AuNps) and polyvinyl butyral (PVB). Electrochemical impedance
                  spectroscopy (EIS) was then used to measure the electrochemical response. It was demonstrated
                  that the biosensor showed different interaction to serum glycoprotein and serum without
                  glycoprotein. The electrodes modified with serum glycoprotein demonstrated higher
                  electron transfer resistances than those modified with BSA, indicating an agglutination
                  reaction on the electrodes. The resulting biosensor detected dengue serum glycoproteins
                  from dengue-infected patients with a detection limit of 80 dilution fold [

                        63

                  ]. In a similar type of study, the serum glycoproteins were detected by a different
                  type of lectin,
                  i.e., Cratylia mollis (CramoLL). However, in this study, a slightly different immobilization
                  method for the lectin was used; CramoLL was immobilized on Fe

3
O

4
 modified gold nanoparticles (AuNps). Notably, this study dealt with three different
                  serotypes of dengue and the developed biosensor showed a wide linear response to various
                  concentrations of dengue serotypes 1, 2 and 3 in sera [

                        64

                  ]. Another study had used bauhinia monandra (BmoLL), another different type of lectin,
                  to detect DENV. Immobilization of this lectin was carried out on a novel gold nanoparticles-polyaniline
                  hybrid composite (AuNPPANI) (
                  Figure 2). The particular biosensor was able to detect and differentiate three dengue serotypes
                  as the viruses have higher electron-transfer resistance than the negative samples,
                  indicating that lectin-glycoprotein complex behaves as the unreactive electron and
                  mass-transfer blocking layer, and prevents the diffusion of ferricyanide toward the
                  electron surface. This lectin-based sensor showed better sensitivity for the detection
                  of dengue serum glycoproteins than a previous study [

                        63

                  ] with a detection limit of 1:150 [

                        65

                  ].

                  Figure 2

Mechanism for the reaction of aniline with chloroauric acid trihydrate and schematic
                        representation of the AuNpPANI-BmoLL-BSA-DEN biosensor system (Retrieved from [

                              65

                        ]).

Apart from lectin, some other studies have employed specific dengue antibody to detect
                  dengue serum glycoprotein. A label-free electrochemical immunosensor was developed
                  on gold electrode, acquired from a recordable compact disk (CD-trode), for the detection
                  NS1 antigen [

                        66

                  ]. Protein A was used for the oriented immobilization of anti-NS1 capture antibody
                  on the gold electrode. Differential pulse voltammetry (DPV) was performed to measure
                  electrochemical response occurred during the interaction between NS1 antigen and anti-NS1
                  antibody immobilized on the electrode. Interestingly, this biosensor was a quantitative
                  assay and was very sensitive with detection limit of 0.33 ng·mL
                  −1 [

                        66

                  ].

Some researchers have exploited more advanced technology/method such as nanotechnology
                  and oriented capture antibody immobilization technique to increase the sensitivity
                  of the biosensor. Since nanomaterial provides extraordinary chemical and physical
                  properties such as large surface per unit mass (50–500 m
                  2/g) and excellent mechanical and electrical properties, these make them particularly
                  useful for electronic detection of biomolecules. In addition, the surface of nanomaterial
                  can be functionalized with appropriate chemical groups so that the desired biomolecules
                  (nucleic acids, enzymes, carbohydrates) can be immobilized [

                        67

                  ]. On the other hand, oriented antibody immobilization method comparatively captures
                  more antigens than random antibody immobilization method, therefore, it is a crucial
                  factor to be considered for improving the sensitivity of the immunoassay. Immobilization
                  of antibodies is considered to be properly oriented and perfectly active when the
                  Fc region, which has no antigen binding affinity, is immobilized on a surface rather
                  than the antigen-binding sites of the antibodies [

                        68

                  ]. In this regard, one specific example is the carbon nanotube (CNT) modified screen
                  printed electrode specific to detect dengue NS1 glycoprotein [

                        55

                  ]. The prominent feature of this immunosensor was the oriented covalent immobilization
                  of anti-NS1 antibody on working electrode, resulting in a better sensitivity of the
                  assay. A much lower detection limit of 12 ng·mL
                  −1 was demonstrated in this study, which is within the NS1 concentration range in dengue
                  infected serum samples. Incorporation of the CNT into carbon ink also proved to be
                  useful and enhances the sensitivity and reproducibility of the immunosensor [

                        55

                  ]. Although this immunosensor showed great sensitivity, its practical application
                  is still debatable as it was not tested on real clinical samples.

Recently, some other studies have developed an electrochemical biosensor for the detection
                  of NS1 in real serum samples [

                        69

                  ,

                        70

                  ,

                        71

                  ,

                        72

                  ]. An electrochemical biosensor using hybrid membrane composed of ConA and lipid membrane
                  has been developed to detect glycoprotein from dengue-infected samples [

                        69

                  ]. Another study has developed similar biosensor but using CramoLL lectin to discriminate
                  glycoproteins from sera of a dengue-infected patient. However, in this study CramoLL
                  lectin was deposited by electrostatic interactions on hybrid nanocomposite composed
                  of gold nanoparticles (AuNps) and polyaniline (PANI) [

                        70

                  ]. Streptavidin/biotin based oriented immobilization method has also been applied
                  to electrochemical immunosensor for the detection of dengue serum glycoprotein in
                  real serum samples. In one study, screen printed carbon electrode was pre-treated
                  with streptavidin/biotin system for the immobilization of the anti-dengue NS1 capture
                  antibody [

                        72

                  ]. Incorporation of this technique resulted in a well-oriented immobilization of the
                  capture antibody and consequently enhanced the sensitivity of the assay with a detection
                  limit of 0.03 µg·mL
                  −1. In terms of versatility, egg yolk immunoglobulin (IgY) has been employed as an alternative
                  biorecognition component to conventional mammalian antibodies for the detection of
                  DENV NS1 protein [

                        71

                  ]. Although, the assay demonstrated a detection limit of 0.09 µg·mL
                  −1 which were higher than the previous study reported by Dias and co-worker [

                        55

                  ], this assay was label free. As mentioned above, NS1 levels vary from 0.04 μg·mL
                  −1 to 2 μg·mL
                  −1 in serum samples of dengue patients [

                        55

                  ]. In this context, a number of developed immunosensors have shown excellent sensitivity.
                  For example, Dias and co-workers reported 0.12 ng/mL while Cavalcanti and co-workers
                  reported 0.33ng/mL limit of detection for their immunosensors, which is lower than
                  7ng/mL and 3.04 ng/mL limit of detection reported in some ELISA-based assays [

                        73

                  ,

                        74

                  ].

Apart from serum glycoprotein, some biosensors have been developed to target other
                  DENV proteins. A chitosan-modified carbon fiber electrode (CFE) was developed to detect
                  DENV envelope protein. In terms of sensitivity, the immunosensor had a better limit
                  of detection for DENV than previously described studies (0.94 ng·mL
                  −1), and a linear range from 1.0 to 175 ng·mL
                  −1 which is clinically relevant to dengue diagnosis. This immunosensor presented an
                  inexpensive assay as the CFE is cheaper than other electrodes such as gold or platinum
                  [

                        33

                  ]. Similarly, DENV type 2 virus has been detected on an alumina-modified platinum
                  electrode [

                        75

                  ]. The main features of this nanobiosensor were short analysis time of 50 min and
                  detection limit of 1 PFU·mL
                  −1 with a linear detection range from 1 to 10
                  3 PFU·mL
                  −1. Additionally, this assay showed no cross-reactivity against other viruses such as
                  chikungunya virus, west nile virus and DENV-3. The reproducibility of the nanobiosensor
                  was acceptable with relative standard deviation (RSD) of 5.9% for triplicate analysis
                  [

                        75

                  ]. In a separate but similar type of development, a label-free immunosensor was developed
                  on a small and thin piece of alumina membrane as the sensing platform for the detection
                  of dengue. Comparatively, this alumina membrane based sensor was rapid with short
                  assay time of 40 min and demonstrated good sensitivity with a detection limit of 0.230
                  PFU·mL
                  −1 [

                        76

                  ].

Besides electrochemical immunosensor, there are also numerous research on the development
                  of electrochemical genosensor for the identification of dengue nucleic acid. Among
                  the reported genosensor is an electrochemical microfluidic biosensor developed for
                  the quantification of target RNA. Interestingly this study designed an inexpensive
                  miniaturized electrochemical detection system (miniEC) and compared its performance
                  to a standard lab-bench electrochemical workstation. The detection mechanism of this
                  electrochemical sensor was based on the hybridization of target nucleic acid with
                  reporter probe DNA conjugated with liposome which interacts with electrochemically
                  active redox species. Upon hybridization, the liposomes were lysed and resulting electrochemical
                  response were measured with miniEC (
                  Figure 3). In terms of performance, this assay showed ten times lower detection limit than
                  the standard lab-bench electrochemical workstation [

                        77

                  ].

                  Figure 3

The principle scheme of the biosensor based on DNA/RNA hybridization, magnetic bead
                        complex formation and fluorescence detection of RNA-specific complexes
                        via intact (
                        a) and lysed (
                        b) liposomes (Retrieved from [

                              77

                        ]).

In another study, a label-free electrochemical genosensor utilizing pencil graphite
                  electrode was developed for the detection of a specific oligonucleotide sequence of
                  DENV Type 1. Hybridization between the probe and the target oligonucleotides was electrochemically
                  monitored through oxidation of guanine. Important features of this genosensor include
                  that ordinary pencil lead was used during preparation of the pencil graphite electrode
                  and simple immobilization technique of the oligonucleotide probe was utilized. In
                  addition, the assay showed a good sensitivity with a detection limit of 0.92 nM and
                  a very short detection time for hybridized probe and its complementary DNA [

                        78

                  ]. Recent developments in genosensor also lead to the detection of particular dengue
                  serotype. In one dengue genosensor, a positively charged gold nanoparticle-polyaniline
                  hybrid composite (AuPANI) was immobilized on gold electrode using its SH-terminal
                  and then negatively charged serotype specific primers were deposited on positively
                  charged AuPANI layer. this assay was highly sensitive as it could discriminate DENV
                  serotypes in the sera of dengue-infected patients at picomolar concentrations [

                        79

                  ]. In addition, nanotechnology based ultrasensitive DNA biosensor has been developed
                  using nanoporous alumina membrane to detect DENV serotype 1 and 3 [

                        80

                  ]. The advantage of using porous nanoelectrode is the high surface area that increases
                  probe loading which in turn enhances the loading of target analytes, and consequently,
                  increases the sensitivity of the assay. Indeed, it was shown that this biosensor has
                  an ultrasensitive detection limit of 9.55 × 10
                  −12 M for the target DNA. Unlike other electrochemical detection methods, where labeled
                  probe and redox species are required for electrochemical signals, this assay only
                  utilizes Fe(CN)

6

                  −4 for DPV based electrochemical signals. The other advantages of this biosensor include
                  its simple preparation and carving of nanostructure which is comparatively easy than
                  other conventional lithography [

                        80

                  ]. In a separate study, a genosensor was developed using integrated sensing membrane
                  based on anodic aluminum oxide membrane. The detection mechanism of this study was
                  quite interesting. First the probe DNA was covalently immobilized within the nanopores
                  of the membrane using a cross-linker. Afterward, the target DNA was deposited within
                  the same membrane. The complementary hybridization of the target DNA to its probe
                  within the pore resulted in blocking of nanopores. This blocking of pores caused the
                  impedance changes and provided detection signals. This study demonstrated that the
                  pore resistance was directly proportional to the concentration of DNA. The genosensor
                  exhibited substantial sensitivity with a detection limit of 2.7 × 10
                  −12 M target DNA analyte. Additionally, this genosensor was capable of discriminating
                  the complementary target sequence from several mismatch to a single base mismatch
                  sequences [

                        81

                  ].

5.2.2. Piezoelectric Biosensor

Piezoelectric biosensor is a mass sensitive detector which utilize mechanically induced
                  changes to produce electrical signals. The transducer used in a piezoelectric biosensor
                  is made of a piezoelectric material such as quartz and the bioreceptor is coated with
                  the piezoelectric material which resonates at a natural frequency. This frequency
                  is controlled by an external potential which produces an oscillating electric field.
                  The biorecognition event causes variation in resonance frequency resulting in current
                  changes which is detected by crystal microbalance (QCM) [

                        62

                  ]. Among the reported piezoelectric biosensor is an immunochip for the detection of
                  DENV NS1 and envelope proteins. This immunochip was developed on QCM and two different
                  monoclonal antibodies for the detection of NS1 and DENV envelope protein were immobilized
                  on two separate immunochips. In addition, a cocktail immunochip containing a mixture
                  of both antibodies was also developed. Three different antibody immobilization techniques
                  were investigated and compared in this study, and protein A was demonstrated to be
                  a better immobilization agent than glutaraldehyde and carbodiimide. The sensitivity
                  of this immunochip assay was 100-fold higher than the traditional sandwich ELISA method
                  while assay time was about one hour. Comparatively, cocktail immunochip demonstrated
                  higher sensitivity than the normal immunochip [

                        82

                  ]. This cocktail immunochip could simultaneously detect NS1 antigen and DENV which
                  circulate at the same time during the acute phase of dengue infection. Similarly,
                  another group of researcher also has designed a piezoelectric biosensor for detection
                  of DENV envelope protein and NS1 glycoprotein [

                        83

                  ]. However, in this study, real serum samples were used for the early diagnosis of
                  dengue. In order to increase the assay sensitivity, various serum pre-treatment methods
                  were investigated and compared. It was found that cibacron blue 3GA gel-heat denature
                  method was the most efficient sample pre-treatment technique with a detection limit
                  of 1.727 µg·mL
                  −1 and 0.740 µg·mL
                  −1 for E protein and NS1 protein, respectively [

                        83

                  ]. A novel biosensing method for piezoelectric biosensor has also been developed.
                  A thin molecularly imprinted polymer specific for nonstructural protein 1, formed
                  by polymerization of monomers, was coated on QCM. These resulting artificial receptors
                  on QCM form amino-acid like cavities that can discriminate epitopes on dengue NS1
                  glycoprotein. This immunochip was shown to be able to detect all four DENV serotypes
                  during the acute phase of dengue infection with a detection limit ranged from 1–10
                  µg·L
                  −1. The artificial receptors used in this particular biosensor may provide valuable
                  alternative to monoclonal antibodies as nonspecific interactions between the test
                  assay and the target protein were minimized when these receptors were used. Another
                  advantage of this assay was that it did not require serum pre-treatment [

                        84

                  ].

In another study, oligonucleotide-functionalized AuNps (also termed as AuNPs probes)
                  were integrated into circulating-flow QCM during the development of piezoelectric
                  genosensor for the detection of DENV serotype 2 [

                        85

                  ]. The assay employed two different AuNps probes which were designated as "mass enhancer"
                  and "sequence verifier" probes. The mass enhancer probe amplified the DENV genome
                  which was then specifically identified by sequence verifier probe resulting in oscillatory
                  frequency change and subsequently detected by QCM as a detection signal. This genosensor
                  was capable of detecting up to 2 PFU·mL
                  −1 of target analyte from dengue-infected sera. This method was quite useful since it
                  does not require expensive equipment and is a label-free assay [

                        85

                  ].

5.2.3. Optical Biosensor

Over the past decades, the optical biosensor has gained a lot of attention due to
                  its high selectivity and sensitivity. Moreover, it is a label-free assay and provides
                  real-time detection. The basic principle of optical biosensor is often based on the
                  adsorption of the target analyte to a selective biorecognition element immobilized
                  on the sensor surface, which causes a change on transducer such as fluorescent signal
                  and measured by a fluorescent detector. Two of the most well-known optical biosensor
                  categories are optical fibers and surface plasmon resonance (SPR) surfaces [

                        62

                  ,

                        86

                  ].

Among the reported optical biosensor is a generic semi-disposable microfluidic biosensor
                  developed to detect RNA of DENV serotype 3 [

                        87

                  ]. In this biosensor, two different DNA probes were used; capture probe which was
                  coupled to supermagnetic beads for the immobilization of target RNA and DNA reporter
                  probe which was conjugated with dye encapsulated liposome for fluorescent detection.
                  Upon interaction with target RNA, both probes hybridized with a target analyte and
                  resulted in liposome-target-bead complex. This biorecognition event was detected by
                  a fluorescence microscopy. The amount of liposomes captured was proportion to the
                  concentration of target RNA and the system had a detection limit of 10 pmol·L
                  −1 of target analyte [

                        87

                  ]. In a similar type of study, a microfluidic biosensor has been developed for the
                  serotype-specific detection of DENV. In contrast to previous studies [

                        87

                  ], which detected only intact dye encapsulated liposome, in this study, both intact
                  and lysed liposome were detected using fluorescence microscopy. This assay showed
                  excellent sensitivity with detection limit of 0.125 nM and 50 pM for intact and lysed
                  liposome detection systems, respectively. This microfluidic biosensor had a short
                  assay time of only 20 min, which was attributed to the presence of dextran sulfate
                  in the hybridization buffer. Moreover, the study showed that this biosensor was capable
                  of detecting all DENV serotypes using four different serotype specific capture probes
                  [

                        88

                  ]. Another group of researchers had developed a serotype specific and sensitive genosensor
                  coupled with isothermal nucleic acid sequence-based amplification (NASBA) to amplify
                  the amount of target RNA [

                        89

                  ]. The important features of this genosensor were that it was portable, inexpensive
                  and easy to use. In this assay, reporter probe coupled with dye-entrapping liposomes
                  assay hybridized with NASBA amplified generic sequence was added to viral RNA. For
                  the generic DENV biosensor, one conserved capture probe was used while for serotype
                  specific biosensor four different capture probes were employed in this study. The
                  amount of liposomes detected in hybridized complex of capture probe-target-reporter
                  probe was equivalent to the concentration of target analyte in the sample. The capture
                  zones were quantified using reflectometer. The result showed that this genosensor
                  could detect as low as 10 PFU·mL
                  −1 of target RNA. However, specificity analysis obtained using serotype specific capture
                  probes showed that the genosensor could discriminate all serotypes of dengue, except
                  dengue serotype 3 [

                        89

                  ].

Serological methods have also been applied in the optical biosensor for the detection
                  of DENV. In this regard, a label-free real-time assay was developed for the detection
                  of dengue specific IgM antibody by using surface plasmon resonance. In this study,
                  the sensing element used was DENV antigen which was covalently bound on gold sensor
                  chip to capture the target analyte IgM antibody from serum. Interestingly, this study
                  employed two immunoassay techniques, direct immunoassay to detect dengue specific
                  IgM antibody and indirect competitive inhibition to detect DENV during acute dengue
                  phase. In a direct immunoassay, when antibody from sera was adsorbed on the sensor
                  with immobilized DENV antigen, an increase in the resonance angle observed. In the
                  case of an indirect immunoassay, dengue specific monoclonal antibody was first incubated
                  with DENV antigen. Later, this antibody and antigen incubated solution was added on
                  the sensor with immobilized DENV antigen, resulting in a decrease of resonance angle
                  which indicated an inhibitory reaction of the immunoassay. This type of biosensor
                  may be quite useful for the diagnosis of both acute and late dengue infection. However,
                  sensitivity and specificity of the assay was not evaluated using real serum samples
                  [

                        90

                  ]. Chemiluminescent measurements are very useful due to their high sensitivity, less
                  background signal, wide dynamic range and comparatively inexpensive instrumentation.
                  One study utilized the combined advantage of chemiluminescent hybridized with the
                  optical fiber sensor [

                        28

                  ]. Similarly, this chemiluminescent optical fiber immunosensor (OFIS) was also developed
                  to detect dengue specific IgM antibody in the clinical serum sample. It was demonstrated
                  that OFIS provided excellent sensitivity as the detection limit was ten times lower
                  than chemiluminescent and 100 times lower than colorimetric ELISA [

                        28

                  ].

To date, the biosensor based assay for the diagnosis of dengue have been developed
                  using different types of transduction mechanisms as well as targeting different analytes
                  and infection stages. In general, the developed biosensors with these various transduction
                  mechanisms are highly sensitive in comparison to conventional methods making it a
                  promising new diagnostic kit for dengue (

                     Table 4
                  ).

viruses-07-02877-t004_Table 4

                  Table 4

Reported LODs and assay times for dengue.

                           Transduction mechanism
                           Analyte (target)
                           Limit of detection (LOD)
                           Assay time (min.)
                           Detection in real sample/spiked sample
                           Ref.

                              Electrochemical

                           RNA
                           0.01 μM
                           –
                           –
                           [

                                    77

                              ]

                           SG
                           80 dilution Fold
                           –
                           serum
                           [

                                    63

                              ]

                           SG
                           1:150
                           –
                           serum
                           [

                                    65

                              ]

                           NS1
                           0.33 ngm·L
                              −1

                           –
                           serum
                           [

                                    66

                              ]

                           NS1
                           0.12 ngm·L
                              −1

                           spiked serum
                           [

                                    55

                              ]

                           SG
                           80-dilution fold
                           –
                           serum
                           [

                                    70

                              ]

                           Viral particle
                           1 PFU·mL
                              −1

                           50
                           –
                           [

                                    75

                              ]

                           Viral particle
                           0.230 and 0.710 PFU·mL
                              −1

                           40
                           spiked serum
                           [

                                    76

                              ]

                           RNA
                           0.92 nM
                           –
                           –
                           [

                                    78

                              ]

                           cDNA
                           9.55 × 10
                              −12 M

                           –
                           serum
                           [

                                    80

                              ]

                           cDNA
                           2.7 × 10
                              −12 M

                           –
                           –
                           [

                                    81

                              ]

                           NS1
                           0.09 µg·mL
                              −1

                           –
                           [

                                    71

                              ]

                              Piezoelectric

                           NS1
                           0.740 µg·mL
                              −1

                           serum
                           [

                                    83

                              ]

                           Envelope protein
                           1.727 µg·mL
                              −1

                           serum
                           [

                                    83

                              ]

                           cDNA
                           2 PFU·mL
                              −1

                           90
                           spiked blood
                           [

                                    85

                              ]

                           NS1
                           1–10 µg·L
                              −1

                           20–30
                           blood
                           [

                                    84

                              ]

                           NS1
                           0.05 μg·mL
                              −1

                           30–60
                           –
                           [

                                    11

                              ]

                              optical

                           RNA
                           10 pmol·L
                              −1

                           –
                           –
                           [

                                    87

                              ]

                           RNA
                           0.125·nM
                           20
                           –
                           [

                                    88

                              ]

                           cDNA
                           100 PFU·mL
                              −1

                           –
                           serum
                           [

                                    89

                              ]

                           IgM antibody
                           1:10
                              6 dilution

                           serum
                           [

                                    28

                              ]

                           IgM antibody
                           12 pg/mm
                              2

                           –
                           blood plasma
                           [

                                    12

                              ]

6. Conclusions

Among the existing conventional methods, virus isolation is of limited use as it is
            laborious, expensive and requires sophisticated lab equipment. Alternatively, reverse
            transcriptase PCR could be used for the detection of dengue viral RNA during early
            stage of infection as it is rapid and sensitive technique although it needs technical
            skills and developed laboratory.

Because of this, serological-based assays are currently the most popular and widely
            used as they are comparatively cheap, sensitive, rapid and have long shelf life reagents.
            Among these serological assays, dengue NS1 ELISA is perhaps a more useful alternative
            to PCR during the acute dengue phase while dengue specific IgM ELISA remains a good
            choice during the convalescent period. Although these conventional techniques are
            very sensitive, they suffer from several drawbacks such as time-consuming, not adaptable
            to real-time detection, require skilled personnel and bulky and expensive machine.
            On the other hand, RDTs for NS1 antigen and IgM antibody detection based on immunochromatography
            are a good alternative to ELISA-based assay since they are easy to use, require no
            equipment and have very short assay time. Although RDTs can provide opportunities
            for point-of-care testing, their lack of sensitivity is a major challenge.

The development of biosensor assays may overcome the limitations of ELISA and RDTs
            since biosensor technology has sensitivity similar or higher than ELISA and with possible
            portability and miniaturization like RDTs. In addition, this technology is easy-to-use,
            inexpensive (since it requires less reagent and low energy consumption), has continuous
            monitoring, and ability to measure the target analyte in complex matrices with minimal
            sample preparation. To date, various transducers and various target analytes have
            been used during the development of sensitive, rapid and/or quantitative DENV biosensors.

However, there is still a long way before commercialization of dengue biosensor could
            be materialized as some issues regarding clinical samples as well as the biosensor
            itself need to be resolved. The clinical samples may require additional preparation
            such as prior RNA extraction before it could be used in genosensor, making it difficult
            to adopt this type of biosensor as a point-of-care test. Meanwhile, integration of
            major biosensor components, including biosensing agent, sample pre-treatment, transducer,
            and detection method into a fully automated yet portable system is a crucial step
            towards the development of commercialized biosensor. For the diagnosis of dengue,
            biosensor could only become alternative commercial assay to conventional tests if
            it could offer rapidity and simplicity equal to RDTs while maintaining the specificity
            of ELISA.

Acknowledgments

Om Parkash is thankful for TWAS-USM postgraduate fellowship and Escience fund (305/PPSP/6113215)
               during this work.

Conflicts of Interest

The authors declare no conflict of interest.

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