Future developments in biosensors for field-ready Zika virus diagnostics
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Since early reports of the recent Zika virus outbreak in May 2015, much has been learned and discussed regarding Zika virus infection and transmission. However, many opportunities still remain for translating these findings into field-ready sensors and diagnostics. In this brief review, we discuss current diagnostic methods, consider the prospects of translating other flavivirus biosensors directly to Zika virus sensing, and look toward the future developments needed for high-sensitivity and high-specificity biosensors to come.
KeywordsZika Flaviviruses Biosensors RT-PCR LAMP Immunoassays
U.S. Centers for Disease Control and Prevention
Emergency Use Authorization
U.S. Food and Drug Administration
Loop-mediated isothermal amplification
IgM antibody capture enzyme-linked immunosorbent assay
Nucleic acid amplification tests
Nucleic acid sequence based amplification
Plaque-reduction neutralization testing
Recombinase polymerase amplification
Reverse transcription polymerase chain reaction
Reverse transcription quantitative real-time polymerase chain reaction
Strand displacement amplification
Reverse transcription-strand invasion based amplification
West Nile virus
Yellow fever virus
Between its first isolation as a zoonotic pathogen in Uganda (1947) and the first major human outbreak of Zika on Yap Island in Micronesia (2007), ZIKV has been primarily observed in Africa and the Pacific [5, 6, 7]. Generally, the flu-like symptoms of infection are mild and include low to moderate fever, headache, joint pain, rash, and fatigue [6, 7, 8, 9, 10]. However, the recent breadth of epidemiological data stemming from the many thousands of cases across South America, the Caribbean, and Central and North America, has uncovered new insights into rare and severe effects on specific subsets of the population. These include a low risk of Guillain-Barré syndrome in adults, and critical risks for pregnant women, including stillbirth, restricted intrauterine fetal growth, and microcephaly [7, 10, 11, 12, 13, 14].
As a member of the Flavivirus genus, ZIKV shares many common genetic sequences and protein structures with other high-interest flaviviruses, including Dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), and Spondweni virus, its most similar relative [15, 16]. On a molecular level, ZIKV features a 10.7 kb single-stranded and positive-sense RNA genome. The polyprotein that this genome encodes cleaves to form several structural proteins, including the envelope (E) and membrane (M) proteins, and nonstructural (NS1 and NS5) proteins . These proteins are the common focus in immunosensing and molecular research for other flaviviruses [12, 13, 16, 17, 18]. Thus, despite the historically limited attention given to ZIKV in the research community, previous work with other flaviviruses may help to inform a rapid turnaround in future ZIKV sensing technologies [7, 8, 15].
In the shadow of the recent epidemic, our understanding of ZIKV pathogenicity has expanded at both the population level and the molecular level. Although several benchtop methods for ZIKV detection have been employed for emergency use, there is still a need for the development and funding of alternative field-ready diagnostic tools. Prompt identification of ZIKV infection at the site of exposure from patient-direct samples is critical in minimizing the global spread of the virus. During the ongoing development and rapid expansion of the ZIKV sensor market, target specificity and sensitivity amidst complex sample matrices are key. In this brief review, we highlight current techniques, emergent diagnostic methods, and considerations for developing future field-ready biosensors.
Gold standards of ZIKV detection
For later stages of ZIKV infection, antibody-based methods can be used. Typically, neutralizing antibodies to ZIKV develop in the human body within the first week of symptoms and continue to remain at detectable levels for up to 12 weeks. During this timeframe, serological assays can be performed to detect the patient’s anti-ZIKV IgM antibodies. However, due to the epidemiological and molecular similarities of ZIKV to other flaviviruses, IgM ELISA assays should be conducted for the antibodies formed against ZIKV, DENV, and CHIKV. In MAC-ELISA, a patient’s sample is added to a well plate pre-coated with antibodies to capture human IgM (Fig. 3b). A virus-specific antigen is then added and washed away, binding specifically to the IgM of infected patients. Finally, an antibody specific to this same viral target that is tagged with an enzyme (e.g. horseradish peroxidase) is added and a chromogenic substrate is used for quantification. Samples from infected patients will thus elicit an optically detectible signal (e.g. absorbance, fluorescence) that may be correlated to IgM concentration. However, the risk of false-positives is high for IgM and IgG assays. If ELISA testing is inconclusive or positive, PRNT should be performed to confirm the presence of ZIKV, specifically .
PRNT typically serves as a secondary test to IgM ELISA and measures the ability of a patient’s antibodies to neutralize a specific virus. In PRNT, serial dilutions of a patient’s serum are added to samples of a viral suspension, and each mixture is applied to a confluent host cell culture (e.g. Vero cells) (Fig. 3c). Following incubation, plaque forming units (PFU) are counted. If neutralizing antibodies specific to this virus are present in the patient’s serum, the associated PFU value will be reduced, and the antibody titer can be determined from the serial dilutions. This method provides better sensitivity and specificity over IgM ELISA, but requires extended time (days), labor, materials, and therefore cost.
Developmental diagnostic methods
Laboratory-based ZIKA assay kits
Alpha Diagnostic International
Recombivirus Human Anti-Zika Virus (ZIKV) Envelope protein IgG/IgM ELISA kits
aTrioplex Real-time RT-PCR Assay
RecombiLISA Zika IgM ELISA kit
DIA.PRO Diagnostic Bioprobes
ZIKV IgG/M ELISA kits
Anti-Zika Virus IgG/IgM ELISA kits
aZIKV Detect IgM Capture ELISA kit
Zika Virus IgM (ZV-IgM) ELISA kit
NovaLisa Zika Virus IgM μ-capture ELISA kit
Lateral flow assay
Chembio Diagnostic Systems
DPP Zika IgM/IgG assay
Anti-Zika Virus IIFT (IgG or IgM)
AccuPower® ZIKV (DEN, CHIKV) Multiplex Real-time RT-PCR Kit
azMAP® MultiFLEXTM Zika RNA Assay
DiaPlexQTM ZCD Detection Kit
TaqMan Arbovirus Triplex Assay (ZIKV/DENV/CHIKV)
aRealStar Zika Virus RT-PCR Kit 1.0
HumqPCR-realtimeTM Zika Detection
Trioplex Real-time RT-PCR Assay
One Step qPCR Detection kit for the Zika Virus
Detection Kit for Zika Virus RNA
aZika Virus Qualitative Real-Time RT-PCR
Zika Virus Real Time RT-PCR
Zika, PCR Kit
Roche Molecular Systems
aLightMix® Zika rRT-PCR Test
Siemens Healthcare Diagnostics
aVERSANT® Zika RNA 1.0. Assay (kPCR)
TaqMan Zika Virus Singleplex Assay
US Biological Life Sciences
Genesig Teal-Time PCR Kit for Zika Virus, Easy
aZika Virus Real-time RT-PCR Test
careGENETM Zika Virus RT-PCR Kit
aZika Virus Detection by RT-PCR Test
Vela Diagnostics USA
aSentosa® SA ZIKV RT-PCR Test
aAptima® Zika Virus Assay
Molecular detection of ZIKV nucleic acid
Reverse transcription PCR (RT-PCR) has become the gold standard for molecular amplification and detection of viruses because of its high selectivity and relatively high sensitivity. Prompted by the 2007 ZIKV outbreak in Yap State, Micronesia, several RT-PCR methods have been developed to specifically identify a multitude of ZIKV strains independent of other flaviviruses. Published ZIKV-specific primer sets target highly conserved regions of the structural membrane (M) and/or envelope (E)  proteins, partial envelope (pE)  protein, or the non-structural (NS1 and NS5) proteins [18, 22, 23, 24, 25]. Detection of ZIKV using RT-PCR has also been shown to work in the presence of many sample matrices including plasma , serum [21, 27], saliva , urine , conjunctival fluid, and semen , thus reducing the need for sample purification or extraction.
Many commercial nucleic acid amplification tests (NAATs) have been developed for ZIKV detection within the past year (Table 1). Between February 26, 2016 and October 21, 2016, the FDA approved ten molecular diagnostic tests for clinical identification of ZIKV under the EUA . Eight of these assays utilize traditional RT-PCR or RT-qPCR amplification (conventional and quantitative real-time thermal cycling) and detection (gel electrophoresis or intercalating dye fluorescence intensity) methods.
The following two non-traditional FDA EUA-approved NAATs claim improved sensitivity, specificity, usability, and speed. The xMAP® MultiFLEXTM assay (Luminex Corp.), uses a proprietary device to complete a series of steps, which include RT-PCR, followed by amplicon-particle hybridization, and final detection via an indicator molecule . The other, Aptima Zika virus assay (Hologic, Inc.), also uses a proprietary device; however, this assay is fully automated and can perform transcription-mediated amplification (exact technique not specified), and qualitative viral detection in the presence of human serum, plasma, or urine, similar to the xMAP® MultiFLEX™ assay . Despite claiming ease-of-use and rapid sample-to-answer times, both methods require approximately 3.5 h and expensive laboratory equipment, and thus laboratory space.
ZIKV biosensors developed in 2016
Range of detection
Limit of detection
Cost per assay
E protein (ZIKV)
NS5 oligonucleotide (ZIKV)
3 fM - 30 pM
E protein RNA (ZIKV)
50-5 × 104 PFU/mL
5 × 103-5 × 106 copies/mL
Of the molecular diagnostic techniques, isothermal genomic amplification has arguably become the most promising method for in-field pathogen identification due to its enhanced specificity, decreased limit-of-detection, reduced assay time, ease of amplification, and number of end-product detection methods. Although not all for ZIKV, several groups have already developed low-cost devices using inexpensive insulating materials (e.g. thermoses) [40, 43, 44] and simple heat-producing elements [45, 46, 47, 48], including non-electrical exothermal reactions [43, 49, 50] (Fig. 4b,c). Many real-time nucleic acid quantification methods have also been used, though again not all for ZIKV, and include measurement of fluorescence , Mg+ pyrophosphate , electrochemical , or colorimetric signal changes, detectable by the human eye [54, 55] or optical sensors [56, 57, 58].
Despite advances in molecular diagnostics, the cost of reagents and equipment and the likelihood of false-negative results present inherent challenges. For these reasons, serological assays remain important alternatives or supplements for detection, particularly when focusing on field-ready assays . These techniques have been most commonly used to detect a patient’s antibody response within a diseased state, as previously described, but can also be extended to direct assays for ZIKV antigens in any sample matrix (i.e. immunoassay), including mosquito pool samples. Flavivirus immunoassays, including ELISA and antibody-based lateral flow assays, have primarily been developed through the antibodies to NS1, NS5, or E proteins . These are also the principal routes of detection in commercial ZIKV MAC-ELISA kits authorized by the FDA (Table 1) .
Depending on the extent of conservation for a targeted epitope among all flaviviruses, some existing assays for DENV or YFV may be adapted directly to ZIKV, but will only have the resolution to broadly identify ZIKV as a flavivirus. Recent immunosensors developed and tested by Cecchetto et al., for example, use impedimetric and capacitive sensing of the NS1 protein from DENV, and have the potential for near immediate conversion to ZIKV detection due to potential cross-reactivity of the anti-NS1 IgG1 antibodies employed  (Table 2). Similarly, flavivirus biosensors have been developed using lab-on-a-chip and lab-on-a-CD technology for label-free optical and electrochemical sensing of DENV through serological IgM or NS1 protein binding [62, 63].
Because ZIKV and other flaviviruses are similarly transmitted to humans by mosquitoes of the Aedes genus, though, the origin of a biomarker detected by a nonspecific immunoassay may be unclear . This potential for cross-reactivity is a primary concern for ZIKV immunosensing and begs further research into high-affinity antibodies with greater species specificity.
In response to these concerns, new research by Dai et al. has focused on discerning the operation of flavivirus antibody recognition for ZIKV through improved characterization of surface protein structures at the angstrom level . Their work has discerned one mode of antibody binding specifically to the ZIKV E protein along a conserved fusion loop, which may be a focal point for future targeted sensors. An extensive survey of E protein structures across 50 ZIKV strains by Badawi et al. has also confirmed multiple conserved epitopes between these, and work by Zhao et al. has revealed several candidate mouse antibodies that demonstrate favorable specificity for ZIKV detection through binding localization at the DIII feature of the ZIKV E protein [59, 66]. However, other proteins may also be ideal candidates for sensing methods. For example, Meltzer et al. have recently highlighted the merits of developing IgM and IgG that are specific to the ZIKV NS1 protein, through which detection may also be more species-specific .
Considerable research is still required to reach our goals of ZIKV sensing across an array of sample matrices with field-ready assay platforms. Fortunately, much has been learned over the past year about ZIKV on a molecular level, and thus many new opportunities have emerged for applying this knowledge toward treatment and diagnostics. Molecular identification of not only ZIKV, but also of other flaviviruses, hinges on the implementation of alternative techniques for amplicon production and detection. Strides have been made in the design of suitable primer sets specific to flaviviruses and ZIKV in particular; however, more development is needed for rapid, in-field detection. For antibody-based assay development, researchers may build their methods from existing cross-reactive assays, but adoption of forthcoming ZIKV-specific antibodies will be necessary for improved specificity. Above all, sensors that can be quickly and inexpensively assembled, screened for quality, and deployed will make the greatest impact in helping to understand and prevent the spread of ZIKV.
Funding for this research was provided by BIO5 Institute, The University of Arizona. AMN acknowledges the support of the SMART scholarship from U.S. Department of Defense. KEM acknowledges support from the U.S. National Science Foundation Graduate Research Fellowship under grant number DGE-1143953. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
Availability of data and materials
Data sharing is not applicable to this article as no primary datasets were generated or analyzed in this work.
AMN and KEM jointly prepared the manuscript and figures with consultation and guidance from JYY. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Figure 4a is reproduced from ref. 33 with permission from the American Chemical Society. Figure 4b is reproduced from ref. 45 with permission from Elsevier. Figure 4c is reproduced from ref. 40 with permission from American Chemical Society. Figure 5a is reproduced from www.zikatest.com with permission from Biocan Diagnostics, Inc. Figure 5b is reproduced from ref. 68 with permission from the authors.
Ethics approval and consent to participate
The need for ethics approval and consent was waived.
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