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A Zika Immunoassay Based on SurfaceEnhanced Raman Scattering (SERS) Nanoprobes Sabrina Alessio Camacho, Regivaldo Gomes Sobral Filho, Pedro Henrique Benites Aoki, Carlos Jose Leopoldo Constantino, and Alexandre G. Brolo ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00639 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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A Zika Immunoassay Based on Surface-Enhanced Raman Scattering (SERS) Nanoprobes Sabrina A. Camacho,†,‡ Regivaldo Gomes Sobral-Filho,‡ Pedro Henrique B. Aoki,‡,§ Carlos José L. Constantino,† and Alexandre G. Brolo ‡,* †

São Paulo State University (UNESP), School of Technology and Applied Sciences, Presidente Prudente, SP, Brazil, 19060900 ‡ University of Victoria (UVic), Department of Chemistry and Center for Advanced Materials and Related Technologies (CAMTEC), Victoria, BC, Canada, V8P 5C2 § São Paulo State University (UNESP), School of Sciences, Humanities and Languages, Assis, SP, Brazil, 19806-900

KEYWORDS: gold shell-isolated nanoparticles, Raman reporter, SERS nanoprobes, Zika detection, cross-reactivity

ABSTRACT: Zika virus (ZIKV) was considered a public health emergency of international concern after the 2015 outbreak. Serological tests based on immunoassay platforms is one of the methods applied on the diagnosis of ZIKV and dengue virus (DENV). However, the high limits of detection (LOD) and the cross-reactivity between ZIKV and DENV are still limitations in immunological tests. In order to tackle these issues, we have designed an immune-specific assay based on surface-enhanced Raman scattering (SERS) nanoprobes. Gold shell-isolated nanoparticles (Au-SHINs) were synthesized with 100 nm Au nanoparticles and 4 nm silica shell thickness coated with Nile Blue (Raman reporter). Then, the SERS nanoprobes were wrapped in a final silica shell and functionalized with monoclonal anti-ZIKV NS1 antibodies. Concentrations of ZIKV NS1 down to 10 ng/mL were probed free of crossreactivity with DENV NS1 antigens.

Zika virus (ZIKV) is a flavivirus that belongs to the same family of dengue (DENV), yellow fever (YF) and Chikungunya.1,2 The first clinical case of Zika fever reported in 2015 in Brazil originated from the Pacific islands.3 Since then, the Zika virus spread across the Americas,4 and an outbreak was declared by the World Health Organization (WHO).3,5 In December 2015, the first warning on the association of ZIKV infection and neurological congenital malformation syndromes was launched by the WHO.6 A definitive correlation between the increasing number of microcephaly cases in newborns and Zika fever was established,7,8 along with the potential of sexual transmission of the virus.9 It was then clear that Zika is a severe threat with much more devastating side effects than other similar diseases, such as dengue. Despite the seriousness of the outbreak, clinical diagnosis of ZIKV infections is still hampered by non-specific symptoms.10,11 The low levels of the Zika biomarkers in human blood also challenge current diagnostic methods based on fluorescent immunological assays. 12 The cross-reactivity of Zika-specific antibodies with other flavivirus highly increases false-positives. Therefore, more accurate diagnostic methods are required in order to increase the sensitivity and to avoid the cross-reactivity, especially between ZIKV and DENV. Motivate by this challenge, we have designed an immunespecific nanoprobe for ZIKV detection based on surfaceenhanced Raman scattering (SERS). The SERS phenomenon has been successfully exploited in different kinds of analytical and biomedical applications.13–19 The nanoprobes used for

shell-isolated nanoparticle-enhanced Raman scattering (SHINERS)20 are ideal for the development of SERS-based immunoassays.21 The ultrathin silica shell (2 - 4 nm)22 prevents the nanoparticles from aggregation and provide a handle for chemical modification.20 In this work, the synthesis of 100 nm Au nanoparticles coated with 4 nm of silica shell is reported. The outer silica layer was modified with Nile Blue (NB) molecules, which act as Raman reporters.23,24 Notice that when the excitation laser (633 nm) coincides with the electronic absorption of NB, the resonance Raman effect also contributes to the overall enhancement. In this special condition, the phenomenon is more appropriately called surface-enhanced resonance Raman scattering (SERRS). The resulting nanostructure was wrapped in a final silica shell to enclose the Raman reporter and to avoid uncontrolled aggregation. The specificity required in immunoassay applications was provided by modifying the outer surface of the nanoprobe with ZIKV antibodies. The SERS nanoprobes reported here enabled the detection of very low concentrations of ZIKV NS1 antigen (10 ng/mL) without crossreactivity with DENV NS1 antigens.

EXPERIMENTAL SECTION Materials The glassware was cleaned with a piranha solution (1:1 ratio of H2SO4/H2O2) and rinsed thoroughly with ultrapure water (resistivity = 18.2 MΩ.cm). All reagents listed below were acquired from Sigma-Aldrich and used without further purifi-

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cation: tetrachloroauric acid (HAuCl4•3H2O, MW = 393.83 g/mol, 99.9%), sodium citrate (C6H5Na3O7•2H2O, MW = 294.10 g/mol, ≥ 99%), hydroxylamine hydrochloride (NH2OH.HCl, MW = 69.49 g/mol, 99.9%), (3-aminopropyl) trimethoxysilane (APTMS, C6H17NO3Si, MW = 179.29 g/mol, d = 1.027 g/mL, 97%), sodium silicate solution (10.6% Na2O and 26.5% SiO2, d = 1.39 g/mL), 3-(triethoxysilyl)propyl isocyanate ((C2H5O)3Si(CH2)3NCO, MW = 247.36 g/mol, 95%), tetraethyl orthosilicate (TEOS, Si(OC2H5)4, MW = 208.33 g/mol, 99.9%), phosphate buffered saline (10x concentrate, suitable for cell culture), nile blue A perchlorate (NB, C20H20ClN3O5, MW = 417.84 g/mol, 95%), N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, C8H17N3.HCl, MW = 191.70 g/mol, ≥ 98%) and Nhydroxysuccinimide (NHS, C4H5NO3, MW = 115.09 g/mol, 98%). The (3-triethoxysilyl) propylsuccinic anhydride (TEPSA, C13H24O6Si MW = 304.41 g/mol, d = 1.070 g/mL, 95%) was purchased from Gelest, dengue virus NS1 (recombinant protein, product code: 8812) from ViroStat Inc and Zika Virus NS1 (recombinant protein product code: AZ 6309) and monoclonal antibody to Zika NS1 (product code: AZ 1225) from Aalto Bio Reagents. Gold Shell-Isolated Nanoparticles (Au-SHINs) Au-SHINs were synthesized following the methodology proposed by Li et al.22 Briefly, 4 mL of Au seeds (~ 40 nm of core size) prepared by citrate reduction25 were diluted in 53 mL of ultrapure water and mixed under stirring, with 0.9 mL of 1% sodium citrate for 3 min and 0.9 mL of 1% HAuCl 4 for another 8 min. The growth of the Au seeds is initiated with the addition of 1.4 mL of hydroxylamine solution (1x10 -2 mol/L). The reaction is completed after 1 h, resulting in AuNPs with ~ 100 nm core size. The AuNPs were then coated with an ultrathin silica shell. 15 mL of AuNPs colloid were diluted in 30 mL of ultrapure water and mixed with 0.5 mL of APTMS (5x10-4 mol/L), under stirring. After 20 min, 2.8 mL of aqueous sodium silicate solution (0.54%, pH = 10) were added and the stirring was kept for 3 more minutes. The mixture was placed in a water bath heating with a controlled temperature (90 – 95°C) from which the Au-SHINs samples were collected after 1 h, centrifuged at 6,000 g for 10 min and redispersed in ultrapure water. This process was repeated at least five times. The silica coating validation for Raman enhancement was performed in experiments with NB solution (concentration: 106 mol/L) and NB solutions containing AuNPs (NB final concentration: 10-6 mol/L) and Au-SHINs (NB final concentration: 10-6 mol/L). Known volumes of these solutions were dropped onto slides, and allowed to dry before the Raman and SERRS measurements. In the experiments with NB solution containing either AuNPs or Au-SHINs (Figure SI1), the fluorescence emission was quenched and NB SERRS spectra were obtained. Surface-Enhanced Raman Scattering (SERS) nanoprobes In a first step, Silica-NB precursor was prepared by mixing 8 mL of NB solution (5x10-3 mol/L) with 10.45 μL of 3(triethoxysilyl)propyl isocyanate (3.8 mol/L), under vigorous stirring for 24 h.26 Then, 250 μL of Au-SHINs (containing an ultrathin silica shell) were diluted in 2.75 mL of ultrapure water + 20 mL of isopropanol and mixed with 9.2 μL of Silica-NB precursor (previously prepared), under stirring for 30 min.27 The ultrathin silica shell allows the metallic nanoparti-

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cles to be covalently bounded to the Silica-NB precursor. In principle, the dye could have been added directly to the gold surface. However, the direct interaction between the dye and the AuNPs can lead to aggregation18. The use of thin silica shell precludes the aggregation problem while allowing a good level of enhancement.20 The final silica shell was assembled by adding 9.2 μL of TEOS (1x10-4 mol/L) and 20 μL of NH4OH (28-30%) into the mixture described above, which was kept under stirring for 4 days. The SERS nanoprobes were centrifuged at 10,000 g for 10 min and redispersed in ultrapure water. This procedure was repeated at least five times in order to obtain clean and concentrated SERS nanoprobes (1x10 12 SERS nanoprobes/mL). The concentration of SERS nanoprobes guarantees an excess of the probes relative to the target. In these conditions, the assay does not depend on the concentration of the probe, but solely on the amount of antigen. Antibody conjugation to the SERS nanoprobes (Ab-ZikaSERS nanoprobes) SERS nanoprobes conjugated with Zika monoclonal anti-NS1 antibodies (Zika-mAb) were prepared according to Li et al.28 with slight modifications. 200 μL of SERS nanoprobes (1x1012 SERS nanoprobes/mL) were redispersed into 200 μL of ethanol and incubated overnight with 1.0 mL of TEPSA (0.12 mol/L). In order to activate the carboxyl terminal groups from TEPSA, the SERS nanoprobes were rinsed with PBS buffer and redispersed into 1.0 mL of NHS (5x10 -2 mol/L) and EDC (0.2 mol/L) PBS buffer solution. After 2h in NHS/EDC PBS buffer, the SERS nanoprobes were rinsed with PBS and 1.0 mL of Zika-mAb (2x10-4 g/mL in PBS buffer) were added for an overnight incubation. Assuming an average footprint of 81.3 nm2 and a molecular weight of ~ 150 kDa for a typical antibody29 and considering that synthesized SERS nanoprobe has an average surface area of 39×103 nm, the antibody coverage on the probe is estimated to be ∼8.3 Zika-mAb/SERS nanoprobes. Unbounded Zika antibodies were removed by centrifugation at 1,500 g for 10 min followed by 3x rinsing with PBS buffer. The resultant SERS nanoprobes (Zika-mAbSERS nanoprobes) were redispersed into 200 μL of PBS buffer for further use. Antibody conjugation to the cover slides Zika monoclonal anti-NS1 antibodies (Zika-mAb) were immobilized onto cover slides following the procedure described by Li et al.30 Cover slides (25 mm length, 18 mm width and 1 mm thick) were cleaned in ultrapure water and ethanol by sonication (10 min each). The slides were incubated overnight in TEPSA solution (0.1 mol/L), rinsed with ethanol and PBS buffer, and then incubated with NHS (5x10-2 mol/L)/ EDC (0.2 mol/L) PBS buffer for 2 h. After that, the slides were rinsed with PBS buffer and punched-well PDMS masks of 5 mm in diameter were sealed onto the slides in order to predefine the regions where different concentrations of Zika NS1 antigen will be further spotted. The immunoassay regions were incubated overnight in Zika-mAb (2x10-4 g/mL) PBS buffer and then in skim milk (1% PBS buffer solution) for 2 h, in order to minimize the non-specific antibody adsorption. SERS immunoassay

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ACS Sensors probe); conjugation onto Zika NS1 monoclonal antibodies (ZikamAb). (b) SERS immunoassay platform for detecting different concentrations of Zika NS1. The platform is irradiated with 633 nm laser line and the SERRS signal from NB molecules, located in a close distance of gold nanoparticles (~ 4 nm), are recorded by area mappings. Brighter spots indicate higher intensity of the NB band at 593 cm-1.

Different concentrations of Zika NS1 antigen (in PBS buffer) were prepared according to clinical relevance for Zika diagnosis.31,32 10 μL of each concentration (from 50 μg/mL to 10 ng/mL) of NS1 in PBS was spotted onto the corresponding well of the immunoassay platform and incubated for 1 h 28. Unbounded antigens were removed by rinsing the wells 3x with PBS buffer. 10 μL of SERS nanoprobes were then dropped onto the immunoassay regions, incubated for 1 h and rinsed with PBS buffer. The scheme of the SERS immunoassay for ZIKV NS1 antigens is presented in Figure 1. Characterization UV-Vis extinction spectra for Au-SHINs and SERS nanoprobes were performed in a Varian spectrophotometer, model Cary 50, from 190 to 1100 nm. Transmission electron microscopy (TEM) images were acquired with a JEOL JEM-1400 transmission electron microscope equipped with a Gatan Orius SC1000 camera. The instrument has a 0.2 nm lattice resolution and a magnification range from x200 to x1,200,000. Resonance Raman and SERRS spectra were carried out with an inVia Raman microscope (Renishaw Inc., Hoffman Estates, IL, USA), laser line at 633 nm, a 50x (NA = 0.75) dry objective (Leica Microsystems, Wetzlar, Germany), 600 lines/mm diffraction grating, 1 s acquisition time, laser power at 17 mW attenuated to 10 %. The background in the SERS spectra was corrected using the software GRAMS AI®, which has a specific background removal tool. The baseline correction was done by manually choosing a certain number of points around the baseline, following by a polynomial fit. The fitted background was then subtracted. The data were also analyzed via a multidimensional projection method known as “Interactive Document Map” (IDMAP).33 This technique is useful for information visualization, where data from a multidimensional space is projected onto a 2D space, creating a plot with maximum preservation of the similarity relationships within the dataset. Details of the method can be found at Paulovich et al.34 IDMAP is a perfect tool to visually demonstrate the degree of separation between an analysis result and its control.

RESULTS AND DISCUSSION The compositions of Au-SHINs and SERS nanoprobes are depicted in Figure 2a. Figures 2b and 2c show TEM images of Au-SHINs and SERS nanoprobes, respectively. The average dimensions of the Au-SHINs and the SERS nanoprobes are given by the size distribution histograms (Figure SI2) built up from the TEM images (Figs. 2b and 2c). The UV-Vis extinction spectra for Au-SHINs and SERS nanoprobes are shown in Figure 2d. The maximum of the localized surface plasmon resonance (LSPR) shifted from 573 nm (Au-SHINs) to 577 nm (SERS nanoprobes) due to the increase on the thickness of silica shell. In shell-isolated SERS, the distance between the metallic nanoparticle and the target molecule can be optimized by tuning the thickness of the silica shell.22 By increasing the shell thickness surrounding the plasmonic nanoparticles, redshifts are observed on the LSPR.35–37 Figure 3 displays the resonance Raman spectrum of NB solution (10-4 mol/L), the SERRS spectra of NB solutions (10-6 mol/L) containing AuNPs and Au-SHINs, and the spectra of the SERS nanoprobes and SERS nanoprobes exposed to 50 μg/mL of Zika antigen onto the immunoassay platform (ZikamAb-SERS nanoprobes). All experiments shown in Figure 3 were performed dropping a known volume of each solution onto slide, let it dry and carried out the resonance Raman and SERRS measurements. The resemblance between the NB SERRS spectra recorded in AuNPs and Au-SHINs confirms that the ultrathin silica shell coating does not affect the spectral characteristics from the NB molecule. An average enhancement factor (EF) of ca. 103 were found for both experiments considering the intensity ratio between SERRS/RR (see SI for details), which is in good agreement to what is predicted by the electromagnetic mechanism.38–40 It is important to emphasize that Figure 3 indicates that the Raman fingerprint from NB was not significantly affected by the chemical modifications used to prepare the Au-SHINs-based SERS nanoprobes. The slight variations in spectral profiles between the SERS nanoprobes and the controls (NB solution, AuNPs + NB solution, and Au-SHINs + NB solution) in Figure 3 confirm that the silica layers, surrounding the NB molecules, did not significantly change the chemical structure of NB. Also, the spectra provided by the SERS nanoprobes are not affected when exposed to 50 μg/mL of Zika antigen onto the immunoassay platform (Zika-mAb-SERS nanoprobes). Both SERRS spectra, from SERS nanoprobes and Zika-mAb-SERS nanoprobes, have similar spectral profile compared to resonance Raman from NB solution (Figure 3). Only small variations in Raman signature are noticeable in Figure 3. For instance, the intensity of the band at 593 cm-1, assigned to phenoxazine ring stretching,41 of the SERS nanoprobes and Ab-Zika-SERS nanoprobes relative to the band at 1640 cm-1, attributed to C–C–C and C– N–C in-plane deformation,42 decreased, which might indicate a preferential orientation of NB molecules within the nanoprobes.

Figure 1. Schematic illustration of (a) Zika-mAb-SERS nanoprobe assembly: Au-SHIN (∼100 nm Au core + 4 nm silica shell thickness); Au-SHIN + NB Raman reporter layer; Au-SHIN + NB Raman reporter layer + final ∼ 10 nm silica shell (SERS nano-


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Figure 2. (a) Cartoon representing the composition of Au-SHINs and SERS nanoprobes. Au-SHIN consists of: 102 ± 8.0 nm Au cores coated with a silica shell of 4.2 ± 1.0 nm thickness; SERS nanoprobe consists of: Au-SHIN + an ultrathin Nile Blue (NB = Raman reporter) layer covalently bounded on the top of the Au-SHIN + a final silica shell with 9.4 ± 1.4 nm thickness, which is subsequently functionalized with Zika anti-NS1 monoclonal antibodies. TEM images recorded for (b) Au-SHINs (102 ± 8.0 nm of core size and 4.2 ± 1.0 of silica shell thickness) and (c) SERS nanoprobes (102 ± 8.0 nm of core size and 9.4 ± 1.4 of final shell thickness). (d) Extinction spectra for AuSHINs and SERS nanoprobes with maximum LSPR at 573 and 577 nm, respectively.

the number of data points can be increased by decreasing the step size in the mappings. However, in that case, the assay time will increase. Thus, a compromise between the assay time and appropriated statistics should be obtained. Despite the 784 spectra collected here, the acquisition time of each mapping was between 20-25 minutes, which is a reasonable time for the diagnostic of the disease into a primary health care setting. The intensity of the band at 593 cm-1 was plotted along the mapped area, where the brighter spots in the map refer to higher intensities in Figure 4. The distribution of intensities in the mapped area indicates that the SERS nanoprobes are fairly dispersed (within 3 μm spatial resolution) along the immunoassay platform surface. Zika NS1 antigens was detected at every probed concentration. The band at 593 cm-1, highlighted in Figure 3, was chosen to analyze the mapping responses, since it is not coincident with any background feature from the control spectra (“blank” and “no Zika antigen” spectra, Figure 4a) and guarantees that the signal observed solely arises from the Zika-mAb-SERS nanoprobes. The control experiments involved: i) Ab-modified cover slides incubated in PBS (“blank” without Zika NS1 antigen and SERS nanoprobes); and ii) Ab-modified cover slides exposed to the SERS nanoprobes without previous incubation in Zika NS1 antigen solutions (“no Zika antigen”). The peak intensities from the band at 593 cm-1 in the SERRS mappings were averaged (from a total of 784 spectra recorded for each concentration of Zika antigen) and a calibration curve was obtained (Figure 4b). A linearity was observed in the semi–log calibration curve over the examined concentration

A discussion about this effect is included as Supporting Information. Another possible explanation to the variation in relative intensities in Figure 3 is related to the formation of the Silica-NB precursor in the SERS nanoprobes. The isothiocyanate groups of 3-(triethoxylsilyl)propyl isocyanate could be covalently attached to the amino terminals of NB. The formation of this new compound (Silica-NB precursor) may be responsible for the slight modifications on the SERS spectral profile of the nanoprobes. In any case, the SERS nanoprobes preserve the unique signature of the dye and they are suitable tools for bio-assays. The immunoassay test, described in Figure 1, was then performed. Glass slides modified with Zika antibodies were incubated with Zika NS1 antigen solutions with concentrations ranging from 50 μg/mL to 10 ng/mL. The slides were then washed and the SERS nanoprobes were added (see Figure 1). The assay was evaluated by SERRS mappings obtained for each concentration, as shown in Figure 4. The SERRS activity observed for this immunoassay is an experimental evidence that the activity of the Zika-mAb was not significantly affected upon the surface modification of the nanoprobe. The mapping technique consists of defining an area in a surface location with a pre-determined number of pixels. SERRS spectra are collected point-by-point from each pixel along the defined area. In the particular case of Figure 4, a 40 μm x 40 μm area was defined in the slide for each concentration of the Zika antigen. SERRS spectra were recorded in 3 μm steps, leading to a total of 784 spectra per antigen concentration. Notice that


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ranges, from 50 μg/mL to 10 ng/mL for the Zika NS1 antigen

(coefficient

Noteworthy is that the lowest concentration of Zika NS1 antigen achieved here (10 ng/mL), although not obtained from biological matrices, is at least four orders of magnitude below enzyme-linked immunoassays (ELISA)-based assay (100 μg/mL) designed for clinically diagnose of Dengue and Zika from human serum.1,43–45 Furthermore, the limit of detection (LOD) of the SERS immunoassay was calculated from the standard deviation of the “blank” response (SDblank) and the slope (b) of the calibration curve (Figure 4b):46 𝐿𝑂𝐷 = 3𝑥𝑆𝐷𝑏𝑙𝑎𝑛𝑘 ⁄𝑏 The LOD (12.5 ng/mL) reached by our SERS immunoassay system is lower than the values found for NS1 antigens (Dengue and Zika). For instance, the LOD reported for Dengue NS1 in sandwich system based on lateral flow strips is 75 ng/mL.32 The higher sensitivity of our system can provide a significant window for early diagnosis of Zika. The LOD improvement is only possible thanks to the enhancement of the NB resonance Raman signal (EF~10 3) enabled by the SERS nanoprobes, highlighting the potential of this platform to drastically increase the sensitivity in acute febrile illnesses (AFIs) diagnostic methods. Compared to other probes, such as fluorescent dyes and quantum dots (QDs), SERS nanoprobes exhibit more advantageous photophysical properties for immunoassay applications. These includes higher sensitivity, higher photostability, and easier quantification. 47–49 The intensity of the SERS signal can be stronger than fluorescent probes, particularly in resonance Raman conditions. 50,51 The narrow bandwidth (~5 nm vs 50 nm for fluorescent dyes) and the shorter lifetime of vibrational excitations (relative to electronic excitations) help in minimizing photobleaching while quenching background fluorescence.52,53 The cross-reactivity frequently found in diagnosing Zika, 1,54,55 was evaluated with 50 μg/mL of Dengue NS1 antigen (Figure 4k). The concentrations of Zika and Dengue antigens in Figures 4c and 4k are the same. However, the Dengue NS1 SERRS mapping (Figure 4k) exhibited the lowest intensities among those maps in Figure 4, showing the specificity of our immunoassay platform towards Zika NS1 antigens. The specificity of SERS-based sandwich immunoassay have also been explored by Sánchez-Purra and co-authors56 for multiplexed detection of Zika and Dengue. Herein, the same antibody (Zika-mAb) were tested for both Zika NS1 and Dengue NS1, which were not a limitation to distinguish the different diseases, reinforcing the specificity observed by Sánchez-Purra et al.56 In order to further demonstrate the distinction ability of our SERS immunoassay when different concentrations of Zika antigen are compared to each other along with a specific concentration of Dengue antigen, one has to resort to statistical or computational methods. Here we confirmed such distinction ability by treating the SERRS mappings (Figure 4) with the IDMAP multidimensional projection technique. IDMAP is a data visualization tool that highlights the similarities (and the differences) present in a dataset. The results of the IDMAP analysis are given in Figure 5. Basically, each circle in the Fig.

5 plot is related to the SERRS intensity at 593 cm-1 within the mapping at a given antigen concentration. The proximity of the circles to each other is an indication of similarity in the SERRS mapping responses. The distinct concentrations of Zika antigen are apart from each other in Figure 5, following a pattern of increasing antigen concentration (right to left). Notably, the specific concentration of Dengue NS1 (50 μg/mL) is well separated from all concentrations of Zika antigen, with exception of 10 ng/mL (closer to the LOD). Moreover, the IDMAP data representation of Dengue NS1 is positioned relatively close to the “no Zika” control. The IDMAP visualization tool in Figure 5 confirms the specificity of our immunoassay towards Zika NS1.Therefore, the SERS platform developed here is capable of detecting different concentrations of Zika NS1 antigen, free of cross-reactivity with Dengue NS1 (from 50 μg/mL to 100 ng/mL).

of

determination

=

0.98762).

Figure 3. Resonance Raman spectrum of 10-4 mol/L NB solution (black); SERRS spectra of 10-6 mol/L NB solutions containing AuNPs (pink) and Au-SHINs (blue); spectra of the SERS nanoprobes (green); and spectrum of Zika-mAb-SERS nanoprobes (red). Excitation laser line at 633 nm. The spectra were background corrected.


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Figure 4. (a) Raman spectra of “blank” and “no Zika antigen”, and SERRS spectrum of Ab-Zika-SERS nanoprobes. (b) Linear dependence between SERRS response (peak intensity from the band at 593 cm-1) and concentration of Zika antigen, from 50 μg/mL to 10 ng/mL. The SERRS response is an average of the SERRS band at 593 cm-1 for each concentration of Zika antigen and the error bars represent the standard deviation (the dataset consisted of 784 SERRS spectra for each concentration). SERRS area mapping (40 μm x 40 μm and step of 3 μm) showing the intensity distribution of the band at 593 cm-1 for (c) - (j) different concentrations of Zika antigen, (k) Dengue NS1 antigen, and (l) “no Zika antigen”. Excitation laser line at 633 nm.


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IDMAP

15 µg/mL 2.5 µg/mL

Dengue NS1 (50 µg/mL)

50 µg/mL 5 µg/mL

500 ng/mL

10 ng/mL

250 ng/mL

No Zika antigen

100 ng/mL Figure 5. IDMAP multidimensional projection for different concentrations of Zika antigen. Each circle in the plot represents the average SERRS intensity at 593 cm-1 within the mapping of a given antigen concentration. The proximity of the circles indicate the similarity between the data (i.e., similar the SERRS mappings responses will lead to circles closer to each other). The average peak intensity from the 593 cm-1-band for a specific concentration of Dengue NS1 antigen (50 μg/mL) and for the “no Zika antigen” control are also represented in the IDMAP projection.

Notes

-

The authors declare no competing financial interest.

CONCLUSIONS ACKNOWLEDGMENT An immune-specific assay based on surface-enhanced Raman scattering (SERS) nanoprobes were successfully applied here for the detection of very low concentrations (10 ng/mL) of Zika virus (ZIKV) antigens. The latter was only possible thanks to the enhancement of the resonance Raman signal (EF~103) that rises from Nile Blue (NB) molecules attached to gold shell-isolated nanoparticles (AuSHINs: 102 ± 8.0 nm Au core and 4.2 ± 1.0 nm of silica shell thickness). ZIKV antibodies bounded to a final silica shell (9.4 ± 1.4 nm) surrounding the SERRS nanoprobes provided specificity to the system towards ZIKV antigens, which was key to decrease the cross-reactivity with dengue antigens and avoid false positives. These experiments highlight the potential of SERS nanoprobes in clinical diagnostic tests.

We acknowledge funding provided by NSERC and FAPESP. We thank Dr. Patrick Nahirney and Brent Gowen for their assistance and access to the TEM.

REFERENCES (1)

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ASSOCIATED CONTENT

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Supporting Information The following files are available free of charge. Silica coating validation for Raman enhancement; Histograms containing the average dimensions of nanoparticles.

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AUTHOR INFORMATION

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Corresponding Author * E-mail: [email protected]

Author Contributions (8)

The manuscript was written through contributions of all authors.

Funding Sources Funding sources provided by NSERC and FAPESP.

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