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

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

Jéssika F.F. Ribeiro, Maria I.A. Pereira, Lara G. Assis, Paulo E. Cabral Filho, Beate S. Santos, Giovannia A.L. Pereira, Claudilene R. Chaves, Gubio S. Campos, Sílvia I. Sardi, Goreti Pereira, Adriana Fontes PII: DOI: Reference:

S1011-1344(18)31159-X https://doi.org/10.1016/j.jphotobiol.2019.03.019 JPB 11487

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

10 October 2018 20 March 2019 26 March 2019

Please cite this article as: J.F.F. Ribeiro, M.I.A. Pereira, L.G. Assis, et al., Quantum dots-based fluoroimmunoassay for anti-Zika virus IgG antibodies detection, Journal of Photochemistry & Photobiology, B: Biology, https://doi.org/10.1016/ j.jphotobiol.2019.03.019

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ACCEPTED MANUSCRIPT Quantum dots-based fluoroimmunoassay for anti-Zika virus IgG antibodies detection Jéssika F. F. Ribeiro1, Maria I. A. Pereira1, Lara G. Assis1, Paulo E. Cabral Filho1, Beate S. Santos2, Giovannia A. L. Pereira3, Claudilene R. Chaves4, Gubio S. Campos4, I.

Sardi4,

Goreti

Pereira3,** [email protected],

Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco,

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Recife, PE, Brazil

Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco,

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2

Recife, PE, Brazil

Departamento de Química Fundamental, Universidade Federal de Pernambuco, Recife,

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3

PE, Brazil

Departamento de Biointeração, Universidade Federal da Bahia, Bahia, BA, Brazil

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4

Fontes1,*

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[email protected] 1

Adriana

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Sílvia

*

Correspondence to: Adriana Fontes, Av. Prof. Moraes Rego, S/N, Departamento de

Biofísica e Radiobiologia, CB, UFPE, 50670-901, Recife, PE, Brazil. **

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Correspondence to: Goreti Pereira, Av. Jornalista Anibal Fernandes, S/N,

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Departamento de Química Fundamental, CCEN, UFPE, 50740-560, Recife, Brazil.

ACCEPTED MANUSCRIPT ABSTRACT Zika virus (ZIKV) has been declared a public health emergency of international concern. ZIKV has been associated with some neurological disorders, and their longterm 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

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immunological response. However, the detection of IgG antibodies can be an important

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approach for ZIKV past infection diagnosis, especially for pregnant women, helping the

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comprehension/treatment of this disease. There has been a growing interest in applying nanoparticles for the efficient ZIKV or antibodies detection. Quantum dots (QD) are

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unique fluorescent semiconductor nanoparticles, highly versatile for biological applications. In the present study, we explored the special QD optical properties to

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develop an immunofluorescence assay for anti-ZIKV IgG antibodies detection. AntiIgG antibodies were successfully conjugated with QDs and applied in a fluorescence

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sensing nanoplatform. After optimization using IgG antibodies, the conjugates were

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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

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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

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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.

Keywords: Semiconductor nanocrystals, Zika Virus, Immunofluorescence assay, IgG antibodies.

ACCEPTED MANUSCRIPT 1. Introduction Zika virus (ZIKV) infections 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

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family that is mainly transmitted by the Aedes mosquito, especially by Aedes aegypti,

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although other transmission pathways have also been discovered, such as by sexual

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contact [3, 4], and blood transfusions [5, 6]. ZIKV has been detected in blood, semen, saliva, and urine. Recent studies showed that consequences of ZIKV infections can

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remain for a long period in the organism, leading to worrisome long-term effects, especially the neurological ones [1, 2, 7, 8].

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Various methods have been proposed and evaluated for Zika diagnostic, including polymerase chain reaction (PCR), IgM-captured immune absorbent assay (MAC-

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ELISA), plaque reduction neutralization (PRNT), and electrochemical biosensing [1, 2,

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9-11]. However, there still exist challenges to overcome, since some methods are rather expensive, time-consuming, laborious, dependent on very specialized equipment, have

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insufficient sensitivity, and/or focus in recent infection diagnosis trough virus or IgM detection [10]. In the search for more efficient methodologies, nano-based materials

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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,

ACCEPTED MANUSCRIPT 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

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appropriated diagnosis. Due to the high sensitivity of fluorescence-based approaches,

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fluoroimmunoassays can be an interesting alternative to develop novel and improved

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detection methods of the virus, as well as antibodies, of Zika [19-21]. Quantum dots (QDs) are fluorescent semiconductor nanocrystals that have been

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widely used as fluorescent probes in biomedical sciences. These nanomaterials possess unique photophysical properties, such as narrow emission and wide absorption spectra,

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size-tunable emission, high photostability, high quantum yields and have chemically active surfaces, which allows their association with biomolecules, providing a specific

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and sensitive labeling tool [22]. Due to these unique properties, QDs showed their

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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 conjugated

with

anti-IgG

antibodies

as

tools

for

an

indirect

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covalently

immunofluorescent nanoplatform, for past infection ZIKV/IgG detection. In this kind of

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analysis, the specificity is firstly given by the interaction of the disease’s antigen (in this case the ZIKV 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, as 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

ACCEPTED MANUSCRIPT 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

2.1 Synthesis and optical characterization of CdTe QDs

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2. Materials and Methods

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Water-dispersed CdTe QDs stabilized/functionalized with 3-mercaptosuccinic

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acid (MSA) were prepared according to a previously published procedure with some modifications [24]. In brief, from metallic tellurium (Te0) and sodium borohydride

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(NaBH4) at pH> 10, and under an inert atmosphere (N2), we obtained an aqueous

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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

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and heating at 90 ± 5 °C for 4 h to allow the growth of nanocrystals for achieving an

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orange emission. QDs were prepared at a molar ratio of 5:1:6 (Cd:Te:MSA). QDs were optically characterized by electronic absorption and emission spectroscopies using a UV–Vis 1800 (Shimadzu) spectrophotometer and an LS55

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spectrometer (PerkinElmer, at λexc = 405 nm), respectively.

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2.2 Preparation of QD-(anti-IgG) bioconjugates CdTe QDs were covalently bound to anti-IgG antibodies [anti-Human IgG (H+L), highly cross-adsorbed produced in goat, from Sigma-Aldrich] as a secondary antibody to be applied in indirect fluoroimmunoassays. For this, the covalent conjugation between carboxyl groups present at the surface of the QDs (1.6 μmol·L-1) to amine groups present in the anti-IgG antibody (46 μg·mL-1) was mediated by the coupling agents N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) at 0.5

ACCEPTED MANUSCRIPT mmol·L-1 (Sigma-Aldrich) and 1.2 mmol·L-1 of N-hydroxysulfosuccinimide sodium salt (sulfo-NHS, Sigma-Aldrich) [24-26]. After, absorption and emission spectra of bioconjugates were acquired to evaluate their optical properties related to bare QDs. Then, the efficiency of this binding was evaluated by the fluorescence microplate assay (FMA)[27]. These assays were performed using the same concentration for bare QDs

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and conjugates.

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The FMA is based on the interaction between proteins and a polystyrene microplate. For this, two negative controls were applied, as follows: C1 – anti-IgG

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antibodies and C2 – bare QDs. It is expected that in FMA both controls show lower

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fluorescence signal when compared to the QD-(anti-IgG) conjugates. The reason for this is that anti-IgG antibodies will be bound to the microplate wells, but their

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autofluorescence signal, under the FMA excitation wavelength, is negligible. On the other hand, non-conjugated QDs do not have an affinity to the microplate wells, thus

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they will be removed during the rinsing steps, also leading to a negligible signal.

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However, when QDs are conjugated with the anti-IgG, they will be bound to the microplate by this protein and an intense fluorescence signal will be detected, even after

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the washings. Then, the better the conjugation, the higher will be the fluorescent signal of QD-biomolecules.

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We incubated 50 μL of all systems in triplicate in polystyrene microplate wells (½ area 96F black microplate- Optiplate HB - PerkinElmer) for 2 h in a humid bath at 37 ºC. Then, we washed the microplate wells three times (3x) with 100 μL of sodium chloride solution at 0.9% (NaCl - from now on named as saline). Afterwards, the samples were analyzed in a fluorescence microplate reader (Wallac Victor2 PerkinElmer). The analysis was performed using the Victor2 software with the excitation band pass filter F405 ± 5 nm and the emission band pass filter F595 ± 30 nm.

ACCEPTED MANUSCRIPT Slits were set as normal and we used 20,000 cw for the lamp. The FMA provided a numerical value proportional to the fluorescence intensity for each system. Since samples were analyzed in triplicate, an average signal was calculated for bioconjugates (“BioconjugatesFL”) and controls (being “ControlFL” related to bare QDs and only proteins). Results were then expressed as relative fluorescence percentages (RFs)

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(

)

Eq. 1

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( )

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calculated by Eq. 1, as proposed by Carvalho et al. [27]:

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2.3 Fluoroimmunoassay based on IgG and QD-(anti-IgG) conjugates Preliminary, assays were performed using IgG antibodies (Sigma-Aldrich) and

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the QD-(anti-IgG) bioconjugates to optimize some detection parameters, such as: (i) the best well sensitization and blocking, (ii) the best bioconjugate amount, and (iii) the

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specificity of the bioconjugates. In order to evaluate the specific interaction between

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conjugates and IgG immobilized in the wells, the activated carboxylic groups of the QDs-(anti-IgG) systems were blocked by using 0.1 mmol.L-1 of TRIS Base for 2 h at 25

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ºC under constant stirring in a Mini Rotator Bio RS-24 (BioSan). The microplate wells were sensitized overnight at 2 – 8 ºC with 50 μL of IgG

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antibody at 2 μg per well. Then, the wells were washed 3x with 100 μL of saline. After that, the blocking of well regions not coated with IgG antibodies was evaluated using non-fat dry milk or bovine serum albumin (BSA, from Sigma Aldrich) diluted at 1% or 5% in saline (100 μL per well), overnight at 2 – 8 ºC. This blocking step was applied to minimize eventual nonspecific interactions between QD-(anti-IgG) conjugates and the microplate well empty sites, as well as to evaluate possible interactions of the conjugates with the blocking agents. The bioconjugate amount per well was also

ACCEPTED MANUSCRIPT evaluated to obtain the highest fluorescence signal for the IgG detection. This test was carried out in duplicate incubating the wells with 50 μL of bioconjugates at 2.0, 1.0, 0.5 and 0.25 g of anti-IgG antibodies by 2 h in a humid bath at 37 ºC. Lastly, we also tested the specificity of the bioconjugate, using an IgM antibody immobilized in the microplate wells in the place of the IgG. For this, we used 50 L of the human anti-A

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monoclonal antibody (clone 9113D10, Lorne Laboratories, Brazil) [28].

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In order to study the possible interferences in the fluorescent signals we used

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three controls, as follows: C3 – IgG/BSA, C4 – BSA/QD-(anti-IgG) and C5 – AntiA/BSA/QD-(anti-IgG), compared with the test that contained IgG/BSA/QD-(anti-IgG).

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At the end of each incubation step, in all experiments, rinsing was performed using saline solution. The signal evaluation was performed in a fluorescence microplate reader

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applying the same conditions described in Section 2.2. All the assays were performed at

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least in triplicate.

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2.4 IgG indirect fluoroimmunoassay for Zika The QD-based indirect fluoroimmunoassay developed tools and protocol were

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then applied for past infection Zika IgG antibodies detection. For this, the ZIKV envelope E protein (MBS596088, MyBioSource) was used as the antigen, and the

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human anti-Zika virus E protein IgG antibody (ZKA64, Ab00779-10.0, Absolute Antibody) was applied to simulate Zika patient’s sample. The immunoreactions were revealed by the QD-(anti-IgG) conjugates developed in Section 2.1. We choose ZIKV E protein, because it is a major viral envelope molecule, involved in receptor binding and membrane fusion. The ZIKV E protein contains different antigenic epitopes that is important targets not only serological assays, but also to produce neutralizing antibodies, and vaccines [29]. Firstly, the microplate was sensitized with ZIKV E protein at 2 g (50 L per

ACCEPTED MANUSCRIPT well), overnight at 2 – 8 ºC, and then washed with saline (3x). The blocking was performed with BSA 5% (100 L per well) incubated overnight at 2 – 8 ºC. After this incubation, wells were also washed with saline 3x. Then, 50 L ZKA64 IgG antibody (2 g per well or 1 g per well) were added and incubated for 2 h in a humid bath at 37 ºC. Finally, the bioconjugates (50 L at 1 g of anti-IgG antibodies per well) were added to

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microplate wells and also incubated for 2 h in a humid bath at 37 ºC.

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For the anti-Zika virus IgG antibody detection, the configuration of the

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microplate well was ZIKV E protein/BSA/ZKA64/QD-(anti-IgG). Two control experiments were also performed: C6 – ZIKV E protein/BSA/ZKA64, considering 2 g

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of antibodies, and C7 – ZIKV E protein/BSA/QD-(anti-IgG). All measurements were performed at least in 3-independent assays in duplicate. After, the ability of the

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bioconjugate detection in human serum (Sigma-Aldrich) enriched with 2 g of antibodies was also tested. The same equipment and the same parameters described in

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shown in Figure 1.

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Section 2.2 were used in these analyses. A schematic representation of this assay is

Figure 1 – Schematic representation of the main steps of QD-based fluoroimmunoassay for anti- ZIKV IgG antibody detection.

3. Results and discussion

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3.1 Characterization of CdTe QDs and bioconjugates A detailed data of QDs and bioconjugates optical characterization can be found in the supporting information. Briefly, both QDs and bioconjugates presented a bright emission in the orange region. The QD’s average size and suspension concentration

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were estimated as 3.1 nm and 7 mol.L-1, respectively.

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3.2 Bioconjugation analysis

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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,

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ca. 3,900% superior to the controls, as indicated in Table 1. The bioconjugation is considered effective when a 100% of RF enhancement is observed, and as higher the

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signal, the better the conjugation procedure [27]. Thus, our results indicated a successful bioconjugation, corroborating a previous study for which CdTe QDs conjugated to IgM

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antibodies had RF about 3,000% and this conjugates recognized, with high specificity,

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red blood cell antigens [28]. We also conjugated successfully green emitting QDs with anti-IgGs (RF = 700%, Supporting Information S02), however we selected orange QDs

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for fluoroimmunoassays, because a higher RF was obtained.

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Table 1. FMA results obtained from the average signals (controls and bioconjugates) and percentage of relative fluorescence (RF) intensity of the bioconjugates. Samples

Fluorescence intensity average (arbitrary units)

RF (%)

C1

336 ± 30

-

C2

302 ± 24

-

12,757 ± 494

3,900

QD-(anti-IgG)

3.3 Optimization of the fluoroimmunoassay

ACCEPTED MANUSCRIPT The optimization of the QD-based fluoroimmunoassay was performed using IgG detection by QD-(anti-IgG) conjugates, also by analyzing the relative fluorescence increase applying FMA. Firstly, the best blocking agent was evaluated and results showed that BSA 5% led to the lowest signal (Figure 2, C4) indicating that there was no significative interaction of bioconjugates directly with the microplate or even with BSA.

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Thus, BSA 5% was used as the well blocking agent in the following assays. Also, the

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molecules did not present a considerable autofluorescence signal (C3).

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Then, QD-(anti-IgG) conjugates (2 g) were incubated in the wells to detect the immobilized IgG, and their fluorescence intensity was compared with the controls. The

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results showed that the wells using the bioconjugates to detect IgGs had a RF of around 2,000% (Figure 2). Furthermore, QD-(anti-IgG) conjugates did not detect unspecifically

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the anti-A antibody (C5 – an IgM antibody), which was immobilized in the microplate,

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confirming the specificity of the bioconjugate to IgG.

Figure 2 – Fluorescence microplate results for IgG detection by QD-(anti-IgG) conjugates (2 g). In C3 – IgG/BSA, C4 – BSA/QD-(anti-IgG) and C5 – AntiA/BSA/QD-(anti-IgG), and the test is IgG/BSA/QD-(anti-IgG).

ACCEPTED MANUSCRIPT After confirming the detection of IgG, using 2 μg of QD-(anti-IgG) conjugates, different concentrations of this bioconjugate (from 0.25 to 2.0 g per well) were used to test the lowest concentration able to effectively detect the immobilized IgG antibody (Figure 3). Results showed an increase in the fluorescence intensity when 1 g of the anti-IgG bioconjugates was used, leading to a high RF of 3,000%, even when compared

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with the amount of 2 g. This can be explained by the prozone effect, which results

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from an excess of antibody impairing the antigen recognition [30]. From these results,

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we observed that concentration between 1 – 2 g of the QD-(anti-IgG) conjugates can be suitable to detect IgG immobilized in a microplate, by fluorescence assays. In this

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way, we could optimize the steps of the indirect QD-based fluoroimmunoassay and we

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proceeded to detect IgG antibodies related to ZIKV past infections.

Figure 3 – Fluorescence microplate results for different concentrations of QD-(antiIgG) conjugates in a sensitized microplate with IgG/BSA 5% (C4).

3.4 QD-based indirect fluoroimmunoassay for Zika QD-(anti-IgG) conjugates were firstly used in fluoroimmunoassays to detect 2 μg/well of anti-Zika virus antibody (ZKA64), using the ZIKV E protein to sensitize the microplate (also 2 μg/well), and an outstanding RF around 8,000% was observed related

ACCEPTED MANUSCRIPT to the C6 control (Figure 4).We also tested the detection of a lower quantity of antiZIKV IgG antibodies (1 μg), which was also detected very efficiently, presenting still a high RF around 3,000% (Figure 4). When compared with control C7, a smaller, but still significant RF signals of, ca. 400% and 110% for 2 μg and 1 μg, respectively, were observed. Although, it was not observed any considerable interaction between the

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conjugates and other proteins such as BSA and anti-A, it seems that conjugates may

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have some unspecific interactions with ZIKV E protein. Despite on that, it was not

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sufficient to compromise the detection, presenting a signal lower than 10,000 arbitrary units (arb. units).

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It has been reported that the ZIKV E protein elicited a response of IgG about 4,500 μg/mL, about 45-fold higher than anti-NS1 IgG [31]. Our QD tools were able to

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detect an amount from 100 to 200-fold smaller than the reported IgG concentration. Thus, the combination of ZIKV E protein as antigen with a QD-based immunoplatform

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may be suitable for anti-E protein IgG detection. In addition, fluoroimmunoassays

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usually present higher sensitivity when compared with the corresponding ELISA assays [32-34]. Moreover, assays based on QD conjugates may be considered less laborious,

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since there is no need of additional reagents, as well as being less susceptible to time and temperature interactions that the ones based on enzymes [35].

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Conjugates were not only able to detect anti-ZIKV IgG antibodies for at least 4 months but also in serum, leading to signals of the same order (ca. 30,000) with a similar sensibility and specificity than the ones reported in Figure 4. As far as our knowledge goes this is the first QD-based fluoroimmunoassay for anti-ZIKV IgG antibodies detection. Thus, our assay can be considered promising for diagnosis of past infections of ZIKV, which could render a practical, simple, and sensitive method.

C6

Test (1 mg)

Test (2 mg)

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C7

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Figure 4 –Anti-ZIKV IgG antibody detection by an indirect fluoroimmunoassay using QD-(anti-IgG) conjugates: C6 – ZIKV E protein/BSA/ZKA64); C7 – ZIKV E

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protein/BSA/QD-(anti-IgG); Test (1 g) – ZIKV E protein/BSA/1μg ZKA64/QD-(anti-

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IgG); Test (2 g) – ZIKV E protein/BSA/2 μg ZKA64/QD-(anti-IgG).

Conclusion

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The main goal of this study was to evaluate a new tool and a potential QD-based

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protocol for anti-ZIKV IgG detection. Herein, QD-(anti-IgG) conjugates were successfully prepared and remained stable, for at least 4 months, allowing their

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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

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with specificity by using the QD-(anti-IgG) bioconjugates. Moreover, the use of ZIKV E protein combined with QD-(anti-IgG) conjugates also led to promising results in the detection of anti-ZIKV IgG antibodies. Thus, fluoroimmunoassays using QDs can be considered a likely approach to diagnosis Zika, allowing the development of practical, simple, and sensitive nanoplatforms to detect anti-ZIKV antibodies. Besides, these QDs bioconjugates can also be used for further diagnosis other arboviruses infections, as well as for monitoring immune response in vaccine trials.

ACCEPTED MANUSCRIPT 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). This work is also linked to the National Institute of Photonics

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(INCT-INFo) and LARNANO/UFPE.

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Disclosure

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

ACCEPTED MANUSCRIPT REFERENCES

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SC

RI

PT

[1] S. Shukla, S.-Y. Hong, S.H. Chung, M. Kim, Frontiers in Microbiology, 7 (2016) 1685. [2] A.M. Nicolini, K.E. McCracken, J.-Y. Yoon, Journal of Biological Engineering, 11 (2017) 7. [3] E. D’Ortenzio, S. Matheron, X. de Lamballerie, B. Hubert, G. Piorkowski, M. Maquart, D. Descamps, F. Damond, Y. Yazdanpanah, I. Leparc-Goffart, New England Journal of Medicine, 374 (2016) 2195-2198. [4] J.M. Mansuy, M. Dutertre, C. Mengelle, C. Fourcade, B. Marchou, P. Delobel, J. Izopet, G. Martin-Blondel, The Lancet Infectious Diseases, 16 (2016) 405. [5] L. Barjas‐Castro Maria, N. Angerami Rodrigo, S. Cunha Mariana, A. Suzuki, S. Nogueira Juliana, M. Rocco Iray, Y. Maeda Adriana, G.S. Vasami Fernanda, G. Katz, F.S.F. Boin Ilka, S.B. Stucchi Raquel, R. Resende Mariângela, L.A. Esposito Danillo, P. de Souza Renato, A. da Fonseca Benedito, M. Addas‐Carvalho, Transfusion, 56 (2016) 1684-1688. [6] D. Musso, T. Nhan, E. Robin, C. Roche, D. Bierlaire, K. Zisou, A. Shan Yan, V.M. Cao-Lormeau, J. Broult, Euro Surveill. , 19 (2014) 20761. [7] D. Musso, C. Roche, T.-X. Nhan, E. Robin, A. Teissier, V.-M. Cao-Lormeau, J Clin Virol, 68 (2015) 53-55. [8] I.N.d.O. Souza, P.S. Frost, J.V. França, J.B. Nascimento-Viana, R.L.S. Neris, L. Freitas, D.J.L.L. Pinheiro, C.O. Nogueira, G. Neves, L. Chimelli, F.G. De Felice, É.A. Cavalheiro, S.T. Ferreira, I. Assunção-Miranda, C.P. Figueiredo, A.T. Da Poian, J.R. Clarke, Science Translational Medicine, 10 (2018) eaar2749. [9] C. Shan, X. Xie, A.D.T. Barrett, M.A. Garcia-Blanco, R.B. Tesh, P.F.d.C. Vasconcelos, N. Vasilakis, S.C. Weaver, P.-Y. Shi, ACS Infectious Diseases, 2 (2016) 170-172. [10] F. Carlo, P. Celia, M. Alfredo, F. Ana Maria Bispo de, V. Antonio Carlos Rosário, R. Bergmann Morais, D. Edison Luiz, T.A.M. Ernesto, S.C. Gubio, F.T.V. Isabelle, L. José Eduardo, S. Luciano Cesar, N. Mauricio Lacerda, B. Michele de Souza, C.S.S. Nathalia, K. Ricardo, M.C.L. Sanny, K. Shirley Vasconcelos, B. Cécile, N.C. Rémi, M.K. Beate, D. Christian, B. Carlos, L. Xavier de, N. Matthias, N. Eduardo Martins, D. Jan Felix, Emerging Infectious Disease journal, 24 (2018) 888-892. [11] D. Acharya, P. Bastola, L. Le, A.M. Paul, E. Fernandez, M.S. Diamond, W. Miao, F. Bai, Scientific reports, 6 (2016) 32227. [12] S.A. Camacho, R.G. Sobral-Filho, P.H.B. Aoki, C.J.L. Constantino, A.G. Brolo, ACS Sensors, 3 (2018) 587-594. [13] B. Zhang, B.A. Pinsky, J.S. Ananta, S. Zhao, S. Arulkumar, H. Wan, M.K. Sahoo, J. Abeynayake, J.J. Waggoner, C. Hopes, M. Tang, H. Dai, Nature Medicine, 23 (2017) 548-550. [14] M.S. Draz, M. Venkataramani, H. Lakshminarayanan, E. Saygili, M. Moazeni, A. Vasan, Y. Li, X. Sun, S. Hua, X.G. Yu, H. Shafiee, Nanoscale, (2018) 11841-11849. [15] M.S. Draz, N.K. Lakshminaraasimulu, S. Krishnakumar, D. Battalapalli, A. Vasan, M.K. Kanakasabapathy, A. Sreeram, S. Kallakuri, P. Thirumalaraju, Y. Li, S. Hua, X.G. Yu, D.R. Kuritzkes, H. Shafiee, ACS Nano, 12 (2018) 5709-5718. [16] O. Adegoke, M. Morita, T. Kato, M. Ito, T. Suzuki, E.Y. Park, Biosensors and Bioelectronics, 94 (2017) 513-522. [17] M.L. Landry, K. St. George, Archives of Pathology & Laboratory Medicine, 141 (2016) 60-67.

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D

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RI

PT

[18] R.K. Singh, K. Dhama, K. Karthik, R. Tiwari, R. Khandia, A. Munjal, H.M.N. Iqbal, Y.S. Malik, R. Bueno-Marí, Frontiers in Microbiology, 8 (2018) 2677. [19] J. Yao, G. Xing, J. Han, Y. Sun, F. Wang, R. Deng, X. Hu, G. Zhang, Journal of Biophotonics, 10 (2016) 657-663. [20] Z. Hu, X. Jing, J. Liu, M. Li, Y. Ye, Y. Chen, Plos One, 11 (2016) e0163682. [21] Z. Zhang, Y. Li, P. Li, Q. Zhang, W. Zhang, X. Hu, X. Ding, Food Chem, 146 (2014) 314-319. [22] M.G.C. Pereira, E.S. Leite, G.A.L. Pereira, A. Fontes, B.S. Santos, Chapter 4 Quantum Dots in: M. Sanchez-Dominguez, C. Rodriguez-Abreu (Eds.) Nanocolloids A Meeting Point for Scientists and Technologists, Elsevier, Amsterdam, 2016, pp. 131158. [23] M. Tang, J. Pi, Y. Long, N. Huang, Y. Cheng, H. Zheng, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 201 (2018) 82-87. [24] P.E. Cabral Filho, A.L.C. Cardoso, M.I.A. Pereira, A.P.M. Ramos, F. Hallwass, M.M.C.A. Castro, C.F.G.C. Geraldes, B.S. Santos, M.C. Pedroso de Lima, G.A.L. Pereira, A. Fontes, Biochim. Biophys. Acta, Gen. Subj., 1860 (2016) 28-35. [25] P.E. Cabral Filho, M.I.A. Pereira, H.P. Fernandes, A.A. de Thomaz, C.L. Cesar, B.S. Santos, M.L. Barjas-Castro, A. Fontes, Int J Nanomedicine, 10 (2015) 4393-4404. [26] C.G. Andrade, P.E. Cabral Filho, D.P.L. Tenório, B.S. Santos, E.I.C. Beltrão, A. Fontes, L.B. Carvalho Jr, Int. J. Nanomed., 8 (2013) 4623-4629. [27] K.H.G. Carvalho, A.G. Brasil Jr, P.E. Cabral Filho, D.P.L.A. Tenorio, A.C.A. de Siqueira, E.S. Leite, A. Fontes, B.S. Santos, J Nanosci Nanotechno, 14 (2014) 33203327. [28] P.E. Cabral Filho, M.I.A. Pereira, H.P. Fernandes, A.A. de Thomaz, C.L. Cesar, B.S. Santos, M.L. Barjas-Castro, A. Fontes, Int. J. Nanomedicine, 10 (2015) 4393-4404. [29] S.N. Slavov, K.K. Otaguiri, S. Kashima, D.T. Covas, Brazilian Journal of Medical and Biological Research, 49 (2016). [30] A.W. Butch, Clin Chem, 46 (2000) 1719-1720. [31] I. Ali, J.P. Chambers, M. Ali, R.C. Tamfu, J Vaccines Vaccin 8(2017) 368. [32] L. Chen, Z. Sheng, A. Zhang, X. Guo, J. Li, H. Han, M. Jin, Luminescence, 25 (2010) 419-423. [33] H. Zhang, T. Xu, L. Gao, X. Liu, J. Liu, B. Yu, Molecules, 22 (2017) 1250. [34] B.A. Robbins, R.M. Nakamura, J Clin Lab Anal, 2 (1988) 62-67. [35] J. Yao, G. Xing, J. Han, Y. Sun, F. Wang, R. Deng, X. Hu, G. Zhang, Journal of Biophotonics, 10 (2017) 657-663.

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

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