Quantum dots-based fluoroimmunoassay for anti-Zika virus IgG antibodies detection

doi:10.1016/j.jphotobiol.2019.03.019

Journal of Photochemistry and Photobiology B: Biology

Volume 194, May 2019, Pages 135-139

https://doi.org/10.1016/j.jphotobiol.2019.03.019 Get rights and content

Highlights

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An ZIKV fluoroimmunoassay using QDs for IgG detection was developed.
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QDs were successfully conjugated to anti-IgG antibodies.
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Conjugates were used in microplates sensitized with ZIKV envelope protein.
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The platform was able to detect anti-ZIKV IgG antibodies in a low concentration.
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Conjugates can be used for diagnosis and monitoring immune response.

Abstract

Zika virus (ZIKV) has been declared a public health emergency of international concern. ZIKV has been associated with some neurological disorders, and their long-term effects are not completely understood. The majority of the methods for ZIKV diagnosis are based on the detection of IgM antibodies, which are the first signs of immunological response. However, the detection of IgG antibodies can be an important approach for ZIKV past infection diagnosis, especially for pregnant women, helping the comprehension/treatment of this disease. There has been a growing interest in applying nanoparticles for efficient ZIKV or antibodies detection. Quantum dots (QD) are unique fluorescent semiconductor nanoparticles, highly versatile for biological applications. In the present study, we explored the special QD optical properties to develop an immunofluorescence assay for anti-ZIKV IgG antibodies detection. Anti-IgG antibodies were successfully conjugated with QDs and applied in a fluorescence sensing nanoplatform. After optimization using IgG antibodies, the conjugates were employed to detect anti-ZIKV IgG antibodies in polystyrene microplates sensitized with ZIKV envelope E protein. The nanoplatform was able to detect anti-ZIKV IgG antibodies in a concentration at least 100-fold lower than the amount expected for protein E immune response. Moreover, conjugates were able to detect the antibodies for at least 4 months. Thus, our results showed that this QDs-based fluoroimmunoplatform can be considered practical, simple and promising to detect Zika past infections and/or monitoring immune response in vaccine trials.

Introduction

Zika virus (ZIKV) infection had a recent outbreak and due to complications associated with this disease, such as microcephaly in newborns and Guillain-Barré syndrome in adults, the World Health Organization listed Zika as a public health emergency of international concern [1,2]. ZIKV is a flavivirus from the Flaviviridae family that is mainly transmitted by the Aedes mosquito, especially by Aedes aegypti, although other transmission pathways have also been discovered, such as by sexual contact [3,4], and blood transfusions [5,6]. ZIKV has been detected in blood, semen, saliva and, urine. Recent studies showed that the consequences of ZIKV infections can remain for a long period in the organism, leading to worrisome long-term effects, especially the neurological ones [1,2,7,8].

Various methods have been proposed and evaluated for Zika diagnostic, including polymerase chain reaction (PCR), IgM-captured immune absorbent assay (MAC-ELISA), plaque reduction neutralization (PRNT) and, electrochemical biosensing [1,2,[9], [10], [11]]. However, there still exist challenges to overcome, since some methods are rather expensive, time-consuming, laborious, dependent on very specialized equipment, have insufficient sensitivity, and/or focus in recent infection diagnosis through virus or IgM detection [10]. In the search for more efficient methodologies, nano-based materials have been increasingly explored to ZIKV or antibodies detection, such as gold nanoparticles [12,13], platinum nanoparticles [14,15] and, quantum dots [16].

The immune response is regulated by the production of IgM and IgG antibodies. IgM antibodies are the first immune response, produced from 4 to 7 days after infection, and can remain in the organism up to three months. Nevertheless, their detection is only trustworthy for 12 weeks after the infection, giving information of recent infections. IgG antibodies are formed a few days after IgMs and remain for several months, allowing the detection of a past infection [17,18]. Zika long-term effects are not completely understood and can lead to further complications, therefore, detection of IgG antibodies can be very useful, especially for pregnant women.

In this context, the search for complementary, reliable, sensitive and, less laborious methods, or even new tools for immunoassays, is still needed to reach an appropriated diagnosis. Due to the high sensitivity of fluorescence-based approaches, fluoroimmunoassays can be an interesting alternative to develop novel and improved detection methods of the virus, as well as antibodies, of Zika [[19], [20], [21]].

Quantum dots (QDs) are fluorescent semiconductor nanocrystals that have been widely used as fluorescent probes in biomedical sciences. These nanomaterials possess unique photophysical properties, such as narrow emission and wide absorption spectra, size-tunable emission, high photostability, high quantum yields and have chemically active surfaces, which allows their association with biomolecules, providing a specific and sensitive labeling tool [22]. Due to these unique properties, QDs showed their potential to improve several diagnostic methods, such as in biosensors and in immunofluorescence assays [19,21,23]. Thus, in this study, we used hydrophilic QDs covalently conjugated with anti-IgG antibodies as tools for an indirect immunofluorescent nanoplatform, for ZIKV past infection/IgG detection. In this kind of analysis, the specificity is firstly given by the interaction of the disease's antigen (in this case the ZIKV envelope E protein) with its captured primary/target antibodies. Nevertheless, the choice of the secondary nanoprobe is also important, not only for the specificity but also for the sensibility of the assay. A highly cross-adsorbed secondary antibody, like the one used in this study, is helpful in eliminating cross-reactivity from other non-target antibodies and proteins. Additionally, an improved and effective conjugate is also prime either to avoid unspecific interactions, from the activated QD's surface groups, as to simultaneously gain sensitiveness. As far as our knowledge goes, this is the first report of the use of QDs for Zika by a fluorescence immunoassay. Furthermore, by modulating the choice of the antigen, this approach could be extended to other flaviviruses, as well as, applied with other ZIKV proteins.

Section snippets

Synthesis and Optical Characterization of CdTe QDs

Water-dispersed CdTe QDs stabilized/functionalized with 3-mercaptosuccinic acid (MSA) were prepared according to a previously published procedure with some modifications [24]. In brief, from metallic tellurium (Te⁰) and sodium borohydride (NaBH₄) at pH > 10, and under an inert atmosphere (N₂), we obtained an aqueous solution of telluride (Te²⁻) ions which were added to a MSA containing cadmium chloride (CdCl₂) solution at pH > 10.5. The reaction proceeded under constant stirring and heating at

Characterization of CdTe QDs and Bioconjugates

A detailed data of QDs and bioconjugates optical characterization can be found in the suplementary data. Briefly, both QDs and bioconjugates presented a bright emission in the orange region. The QD's average size and suspension concentration were estimated as 3.1 nm and 7 μmol.L⁻¹, respectively.

Bioconjugation Analysis

According to the bioconjugation analysis by FMA, a weak signal of the controls C1 and C2 was observed, while the conjugates presented high fluorescence intensity, ca. 3900% superior to the controls, as

Conclusion

The main goal of this study was to evaluate a new tool and a potential QD-based protocol for anti-ZIKV IgG detection. Herein, QD-(anti-IgG) conjugates were successfully prepared and remained stable, for at least 4 months, allowing their application in fluoroimmunoassays. The use of IgG immobilized in a microplate allowed us to optimize the assay steps and this antibody was detected efficiently and with specificity by using the QD-(anti-IgG) bioconjugates. Moreover, the use of ZIKV E protein

Acknowledgments

This work was supported by the Brazilian agencies: Coordenação de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and, Fundação de Amparo a Ciência e a Tecnologia do Estado de Pernambuco (FACEPE, for J.F.F. Ribeiro scholarship and grant APQ-0152-2.09/16). This work is also linked to the National Institute of Photonics (INCT-INFo) and LARNANO/UFPE.

Disclosure

The authors confirm that there are no conflicts of interest in this work.

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Quantum dots-based fluoroimmunoassay for anti-Zika virus IgG antibodies detection

Highlights

Abstract

Introduction

Section snippets

Synthesis and Optical Characterization of CdTe QDs

Characterization of CdTe QDs and Bioconjugates

Bioconjugation Analysis

Conclusion

Acknowledgments

Disclosure

Lancet Infect. Dis.

J. Clin. Virol.

Biosens. Bioelectron.

Food Chem.

Spectrochim. Acta A Mol. Biomol. Spectrosc.

Biochim. Biophys. Acta, Gen. Subj.

Front. Microbiol.

J. Biol. Eng.

N. Engl. J. Med.

Transfusion

Euro Surveill.

Sci. Transl. Med.

ACS Infect. Dis.

Emerg. Infect. Dis. J.

Sci. Rep.

ACS Sensors