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Gold NanoBipyramids Performing as Highly Sensitive Dual-Modal Optical Immunosensors Andreea Campu, Frederic Lerouge, Denis Chateau, Frederic Chaput, Patrice Baldeck, Stephane Parola, Dana Maniu, Ana-Maria Craciun, Adriana Vulpoi, Simion Astilean, and Monica Focsan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01689 • Publication Date (Web): 15 Jun 2018 Downloaded from http://pubs.acs.org on June 15, 2018

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Analytical Chemistry

Gold NanoBipyramids Performing as Highly Sensitive Dual-Modal Optical Immunosensors Andreea Campu†§, Frederic Lerouge‡, Denis Chateau‡, Frederic Chaput‡, Patrice Baldeck‡, Stephane Parola‡, Dana Maniu§, Ana Maria Craciun†, Adriana Vulpoi#, Simion Astilean†§, Monica Focsan*† Nanobiophotonics and Laser Microspectroscopy Center, Interdisciplinary Research Institute on BioNano-Sciences, Babes-Bolyai University, Treboniu Laurean No.42, Cluj-Napoca 400271, Romania †

Ecole Normale Superiéure de Lyon, CNRS, Université Lyon 1, Laboratoire de Chimie UMR 5182, 46, allée d'Italie, F-69364, Lyon Cedex 07, France

‡

Biomolecular Physics Department, Faculty of Physics, Babes-Bolyai University, M Kogalniceanu No. 1, Cluj-Napoca 400084, Romania §

Nanostructured Materials and Bio-Nano-Interfaces Center, Interdisciplinary Research Institute on BioNano-Sciences, Babes-Bolyai University, Treboniu Laurian No. 42, Cluj-Napoca 400271, Romania #

* Corresponding Author: [email protected] (Monica Focsan)

ABSTRACT

In this work, we demonstrate the feasibility of gold bipyramidal-shaped nanoparticles (AuBPs) to be used as active plasmonic nanoplatforms for the detection of the biotin-streptavidin interaction in aqueous solution via both Localized Surface Plasmon Resonance and Surface Enhanced Raman Scattering (LSPR/SERS). Our proof of concept exploits the precise attachment of the recognition element at the tips of AuBPs where the electromagnetic field is stronger, which is beneficial to the surface sensitivity of longitudinal LSPR on the local refractive index, and to the electromagnetic enhancement of SERS activity too. Indeed, successive red shifts of the longitudinal LSPR associated with increased local refractive index reveal the attachment of para-aminothiophenol (p-ATP) chemically labelled Biotin to the Au surface and the specific capture of the target protein by biotin-functionalized AuBPs. Finite-Difference Time-Domain simulations based on the reconstructed index of refraction confirm LSPR measurements. However, the molecular identification of the biotin-streptavidin interaction remains elusive by LSPR investigation alone. Remarkably, we succeeded to complement the LSPR detection with reliable SERS measurements which permitted to (a) certify the molecular identification of biotin-streptavidin interaction and (b) extend the limit of detection of streptavidin in solution toward 10-12 M. Finally, to further probe the possibility to implement the AuBPs as dual LSPR-SERS based immunoassays in solution for real clinical diagnostics, we additionally investigated the AuBP’s performance to transduce the specific antihuman IgG- human IgG binding event, providing thus a reference design for building unique plasmonic immunoassays for dual-optical detection of target proteins in aqueous solution. Keywords: dual detection, colloidal immuno-sensor, Gold Bipyramids, LSPR, SERS detection

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Early detection of specific disease biomarkers has gathered more and more attention since the presence and the progression of a disease involves changes at their molecular level in the body.1 Specifically, once the specific antibody-antigen recognition interaction -as primary step in immunoassayshas been realized, the biological event has to be transformed into signals, which should be easily detected and recorded for rapid and real-time diagnostic applications. Currently, enzyme-linked immunosorbent assay (ELISA) is the most widespread tool to detect the presence of an antigen or its complementary antibody.2,3 The major limitation of ELISA consists in its moderate detection sensitivity, being able to detect the concentration of cancer biomarkers in clinical serum only after the critical level has been reached. Beside this technique, other complex readout tools used for protein biomarkers determination are fluorescence, radio-immunoassay, Western blot, mass spectrometry or electrochemical detection.4 But, although the fluorescence, for example, has extensively been employed in immunoassay labelling considering its inherent sensitivity, the photobleaching process may complicate the data interpretation. Moreover, the simultaneous detection of multiple biomarkers with this technique becomes impossible, being limited by the broad spectral width of fluorescence response. Therefore, simple, affordable, and accurate point-of-care (POC) methods resistant to photobleaching and with improved sensitivity and selectivity are in high demand in the field of clinical diagnosis. Significant research has been currently dedicated to design innovative and efficient optical biosensors based on plasmonic transducers, including Localized Surface Plasmon Resonance (LSPR) or Surface-Enhanced Raman Scattering (SERS).5–8 Thanks to the progress of nanotechnology, colloidal gold nanoparticles (AuNPs) have drawn considerable interest as attractive plasmonic transducers, because of their unique physical and optical properties, which make them excellent scaffolds for the development of biosensors for a variety of target analytes. Lately, this enthusiasm has been directed towards anisotropicshaped AuNPs, which exhibit at their tips and edges a locally enhanced-electromagnetic field that unlocks a variety of new strategies to improve and enlarge the implementation domain of AuNPs as low-cost homogeneous immunosensors in aqueous solution.9 Additional to their strong signal intensities and finely tunable surface chemistry, the high surface area to volume ratio allows these nanomaterials to be regarded as promising for biochemical detection. In particular, LSPR phenomenon -which is the result of the collective oscillations of the metal conduction electrons after being exposed to a light beam, is strongly dependent on the size and shape of the AuNPs as well as their interparticle spacing and the dielectric microenvironment surrounding them.10–15 The last property is currently exploited to detect molecular targets of interest upon their binding to the surface of the NP.8,16,17 Concretely, when a biomaterial is immobilized on the surface of AuNPs, any change due to mass accumulation is accompanied by a refractive index change, which can be directly monitored by the LSPR spectral response, affording thus the basis for LSPR-based biosensors.8 Consequently, the high demand for LSPR biosensing devices has 2 ACS Paragon Plus Environment

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increased lately, being considered now a leading technology for the label-free, real-time detection and the study of biological binding events.18 Despite high sensitivity in detecting the amount of biomaterial attached on the surface, the structure and the molecular identification of the immobilized target biomaterial is elusive in LSPR-based sensors. The excitation of LSPR enhances the local electromagnetic field promoting SERS. Furthermore, the geometry and size of the AuNPs can be tuned such that their characteristic LSPR response match the Raman excitation wavelength, offering thus a higher light scattering enhancement and consequently an increased SERS probe immunoassay sensitivity.19,20 Moreover, SERS-based biosensors are known to be able to detect analyte molecules of interest at very low concentrations with extraordinary specificity offered by Raman spectral fingerprinting abilities.21,22 Additionally, compared to other conventional detection techniques, SERS has the advantage to be used for sensitive immunoassay development because of its excellent multiplexing detection capability and ease-to-use without complicated sample preparation. In order to traduce antigen-antibody binding events that resulted from different immunological reactions, several efficient transduction strategies -in particular based on anisotropic AuNPs, have been implemented in the literature during the last decades. For example, Caswell et al. have demonstrated that biotin-functionalized gold nanorods (AuNRs) can be easily end-to-end linked upon the addition of streptavidin molecules.23 Later, Wang et al. brought a significant contribution to the field of immunosensors with real applicability in early diagnosis by designing a novel plasmonic biosensor based on AuNRs to detect the hepatitis B surface antigen. By monitoring the LSPR shift response of AuNRs induced by the immunological reaction, a limit of detection of 0.01 IU/mL was achieved.24 Recently, we have also proposed a new strategy to label streptavidin onto the AuNRs surface in order to improve the sensitivity of LSPR shift induced by biotin-streptavidin interaction. By employing AuNRs as amplification labels, the LSPR response of the biotin-functionalized LSPR biosensor was 26-fold enhanced compared to the free streptavidin detection.25 Although AuNRs were initially preferred to be employed as anisotropic-based biosensors, theoretical simulation and experimental results have recently proved that gold nanobipyramids (AuBPs) are more chemically stable compared to AuNRs, exhibit higher refractive index sensitivity (RIS) as well as figure of merit (FOM) values, and -more importantly- the local field enhancement is several times larger because of their two sharp tips.26–28 This last important property can be regarded as key aspect for intrinsic “hot-spot” generation, improving target analyte detection via SERS. Concretely, Li et al. have compared the SERS performances between these two types of elongated plasmonic NPs, demonstrating thus the superiority of AuBPs by employing 4mercaptophenol as Raman-active probe.28 Additionally, the possibility to obtain a fine tuning on the geometry, shape and truncation of the synthesized AuBPs, controlling precisely the longitudinal LSPR response between 600 and 2000 nm, was proved by Parola’s group.29 Moreover, Xu et al. have recently 3 ACS Paragon Plus Environment


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demonstrated the ability of highly uniform AuBPs for the ultrasensitive colorimetric detection of H5N1 virus, achieving a limit of detection of 1 pg/mL.30 Despite all these fascinating plasmonic properties, only a few articles have reported the development of AuBPs-based nanosensors for immunoassay applications,31 and no one made use of simultaneous integration of LSPR and SERS sensing on AuBPsbased nanosensors in aqueous solution. In fact, dual mode detection could provide a real advantage over the direct detection by LSPR or SERS alone. Concretely, in real sensing situations SERS measurements are not self-sufficient to provide the full information about the binding event or analyte and need to be combined and complemented with LSPR measurements. The complementarity of LSPR spectral-shift assays is in fact relevant to a wide range of molecules which are not efficient SERS scatters or exhibit overlapping peaks due to structural similarities and therefore their presence should be differentiated by LSPR shifts. Also, the use of the LSPR method is still highly suitable in several SERS sensing situations because it allows a fast response and makes the monitoring of kinetics possible. In this context, it is of high interest to efficiently integrate the functionalities of both LSPR and SERS sensing onto the same AuBPs and further explore its potential as innovative immunosensor. Herein, we develop for the first time a novel synergistic LSPR/SERS-active immunosensor in aqueous solution based on gold bipyramidal-shaped nanoparticles, which is able to demonstrate and highlight the specific biotin-streptavidin interaction as a “proof-of-concept”. The novelty relies in the chemically formed linkage between the biotin and a well-known Raman reporter probe (herein pAminothiophenol, p-ATP) in order to form an active p-ATP-biotin system, which will be covalently grafted on the AuBPs surface, avoiding thus further activation steps. Subsequently, the as-obtained Raman-labelled biotin complex, as recognition element, was attached at the tips of AuBPs where the intrinsic electromagnetic hot spots are stronger, this aspect being beneficial to the sensitivity of longitudinal LSPR on the local refractive index and to the electromagnetic enhancement of SERS activity too. Specifically, while the streptavidin detection by biotin-functionalized AuBPs is demonstrated by successive red shifts of the longitudinal LSPR associated with the increase of the local refractive index, also supported by FDTD numerical calculations, the SERS technique -due to its fingerprinting capabilities, proves the specific capture of the target protein by simultaneous molecular identification of the characteristic Raman bands of p-ATP at 1079 and 1591 cm-1 together with the appearance of the specific bands of the biotin-streptavidin interaction. Remarkably, benefiting of the by reliable SERS technique, we were able to extend the limit of detection (LOD) of streptavidin in solution toward 10-12 M by monitoring the recognition interaction via the band at 1468 cm-1. We have purposely performed our demonstration with a class of AuBPs with high SERS activity selected by optimizing the LSPR position relative to laser line at 785 nm, using the p-ATP as SERS probe. To further demonstrate the possibility to implement the AuBPs as dual LSPR-SERS based immunoassays in solution for real clinical diagnostics, 4 ACS Paragon Plus Environment

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we additionally investigated the AuBP’s performance to transduce the specific anti-human IgG- human IgG binding event. To note that, until now, SERS has not been implemented in combination with LSPRbased sensing in immuno-analysis using AuBPs as optical transducers in aqueous solution. Our results indicate that the as-designed dual plasmonic nanoplatform can represent an excellent optical detection platform in aqueous solution of relevant biomarkers in the future. EXPERIMENTAL SECTION Preparation of the Raman label of the recognition element. Commercially available Biotin-NHS (100 mg, 0,318 mmol) was dissolved into acetonitrile and dry DMF. A solution of p-ATP (36 mg, 0,318 mmol) in acetonitrile was then added dropwise under nitrogen atmosphere with mild stirring. After the addition of a slight amount of triethylamine the mixture was left undisturbed overnight.32 After treatment by evaporation and precipitation, the obtained white powder was dissolved in methanol for further use. The biosensing protocols were further conducted with a p-ATP activated biotin solution of 15 mM. This resulting p-ATP activated biotin was denoted as p-ATP@Biotin later. Biosensing protocol. Firstly, to test the LSPR and SERS sensitivity of the synthesized AuBPs, 1 mL AuBPs solution with different aspect ratios ranging from 2.90 to 5.79 were incubated with 5 µL of p-ATP ethanoic solution of 10-4 M for 30 minutes. The proposed biosensing protocol and the coupling of the obtained most efficient bipyramids in terms of sensitivity for both SERS and LSPR with activated streptavidin are shown in Scheme 1 and Figure S1. Firstly, the p-ATP@Biotin was grafted on the surface of the AuBPs by the addition to the colloidal gold solution followed by heating 30 minutes at 45 ºC. The mixture was then left undisturbed for 24 h. The new hybrid nanoparticles (denoted as p-ATP@BiotinAuBPs) were used as LSPR-SERS nanoplatforms for the specific detection of streptavidin. The specific binding to p-ATP@Biotin-AuBPs was conducted by incubation with target protein overnight at room temperature. In order to determine the LOD of the as-designed nanoplatform, different streptavidin concentration solutions (i.e. between 10-6 M and 10-12 M) were tested.

Scheme 1. Schematic illustration of the biosensing protocol steps employed for the development of the plasmonic nanosensor.

Then, with the aim to demonstrate the possibility to implement the AuBPs as colloidal immunosensor for real clinical diagnostics, we also probe the specific anti-human IgG- human IgG 5 ACS Paragon Plus Environment


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binding event. Concretely, for the human IgG detection, the as-synthesized AuBP were firstly purified at 8000 rpm for 15 minutes. The immobilization of the anti-human IgG was achieved by exposing the AuBPs to 10 mM p-ATP molecules, which bind spontaneously to the Au surface with their thiol group, and GA molecules, which are the crosslinkers between the amine groups of the p-ATP and the amine moieties of the anti-human IgG.33 To note that the Raman reporter excess was removed under the same centrifugation conditions, the sediment was redispersed in 5% GA spacer and the mixture was left undisturbed for 1 h. Afterward, the anti-IgG antibody 0.5 mg/ml was added and left to react with the aldehyde groups modified p-ATP@AuBPs for 2 h. The unbound molecules were extracted by centrifugation and the SERS probes were redispersed in ultrapure water, the non-specific adsorption was avoided using 1% BSA (in PBS). Finally, human IgG 1 mg/ml was added for detection.

RESULTS AND DISCUSSION Spectroscopic characterization of Raman labeling of the recognition element. In order to endow the recognition element with chemical affinity to AuBPs, we proceed with the molecular conjugation of Biotin with p-ATP -a small molecule with strong affinity to the Au surface via thiol group. The molecular conjugation and structure of the new active p-ATP@Biotin recognition compound were characterized the by FT-IR spectroscopy (see schematic conjugation in Figure 1A and Figure 1B-solid spectra). Quantum chemical calculations were additionally performed to assign accurately the vibrational frequencies of pATP and p-ATP@Biotin. The assignment was assumed by direct comparison between the experimental and calculated IR spectra taking in consideration both the frequency sequence and the intensity pattern as well observation of normal modes animated by using GaussView 5.0 program (the optimized geometries of the p-ATP and p-ATP@Biotin with the symbol of the atoms, and corresponding simulated IR spectra are given in Figure S2 and Figure 1B-dotted spectra, respectively). The most important experimental and calculated vibrational IR frequencies together with the proposed assignments are summarized in Figure 1C (for a detailed assignment see the Supporting Information).


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Figure 1. (A) Schematic representation of the conjugation of NHS-Biotin with p-ATP in order to endow chemical affinity to the gold core; (B) DFT Simulation and experimental FT-IR spectra of p-ATP (spectra a and c) and p-ATP@Biotin (spectra b and d); (C) Assignment of the major IR bands in both simulated and experimental spectra of p-ATP and p-ATP@Biotin, respectively. Optical and morphological characterization of the synthesized AuBPs. Figure 2A presents the extinction spectra of different aspect ratios of the as-synthesized AuBPs in aqueous solution. All spectra exhibit the typical signature of transverse and longitudinal LSPRs.29 The spectral position of the transversal LSPR band at approximately 510 nm and the large tunability of longitudinal LSPR band from 636 nm to 920 nm are correlated with their aspect ratios, ranging from 2.9 to 5.79. The formation of AuBPs was also confirmed by TEM observation showing typical diamond-like structures. Figure 2B shows a representative TEM image of the selected AuBPs with the longitudinal LSPR band located at 793 nm. Moreover, according to the TEM analysis realized using an image processing Image J toolkit, the average length x width dimension is 86 nm x 28 nm (Figure S3A-B). The presence of positive CTAB surfactant bilayers on the surface of AuBPs is confirmed by zeta potential of +32 mV (ζ-deviation = 4.02) (see Figure S3C).



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Figure 2. (A) Extinction spectra of the synthesized AuBPs with different aspect ratios ranging from 2.9 to 5.79; (B) Representative TEM image of the selected AuBPs with the longitudinal plasmon band located at 793 nm.

Investigation of SERS and LSPR sensitivity as function of AuBPs’s aspect ratios. In the following we exploit the optical response tunability of the AuBPs in combination with the strong electromagnetic enhancement at their tips to operate them as efficient dual modal LSPR-SERS nanoplatform in solution, this aspect being of great importance for achieving efficient high-throughput biodetection. Concretely, for a more reliable biodetection, we would like to select the most powerful class of AuBPs in terms of sensitivity for both SERS and LSPR. For this purpose, the SERS efficiency of the as-synthesized AuBPs with different aspect ratios was firstly investigated using p-ATP as Raman analyte molecule. To note that the used concentration of p-ATP was 10-4 M and was kept constant for all AuBPs. p-ATP is considered an ideal target Raman analyte due to its thiols which interact very strongly with the Au atoms on the Au surface via back π-bonding.34 Specifically, the SERS performances of the synthesized AuBPs were tested using a 785 nm excitation laser from a portable Raman spectrometer. As a first observation, all the pATP-grafted AuBPs were found to be SERS active (Figure 3A) and characteristic bands of p-ATP molecules were recorded in all cases. In particular, the most intense vibrational modes arising in the spectrum are the C-S stretching vibration at 1079 cm-1 and the C-C stretching vibration at 1585 cm-1.35 The enhancement of these two specific Raman bands is explained by the electromagnetic effect, representing a direct proof that p-ATP molecules are covalently grafted on the tips of AuBPs. Interestingly, the SERS efficiencies are different between each AuBP’s aspect ratios and the bands


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intensities vary with the LSPR localization of the AuBPs. The marked spectrum in Figure 3A clearly reveals that the AuBPs with longitudinal LSPR at 793 nm exhibits the best performance in terms of SERS sensitivity under excitation at 785 nm, confirming their great potential to be further used as efficient SERS biosensing nanoplatforms.

Figure 3. (A) SERS spectra of p-ATP grafted on AuBPs surface (denoted as p-ATP@AuBPs) presented in Fig. 2A using 785 nm excitation laser. The dotted spectrum represents the simulated normal Raman spectrum of p-ATP; (B) Normalized extinction spectra of AuBPs before (solid spectra) and after (dotted spectra) the adsorption of p-ATP molecules. Furthermore, we evaluated the LSPR sensing performance of the AuBPs by monitoring the dependence of the surface RIS as a function of LSPR peak wavelengths. For this, we grafted p-ATP molecules at constant concentration of 10-4 M on the surface of the synthesized AuBPs with different aspect ratios. Figure 3B illustrates the extinction spectra of AuBPs before and after grafting with p-ATP molecules, revealing a red-shift of the longitudinal LSPR band varying between 8 and 12 nm in the maximum wavelength position for all synthesized AuBPs, while the transversal LSPR band displays almost no detectable modification in position. This phenomenon can be explained by the RI modifications, as a consequence of capture of p-ATP analyte, only within a confined area close to the sensing surface, more exactly at the tips of AuBPs where the electromagnetic field is stronger (more details in Supplementary Information). Proof-of-concept for dual LSPR - SERS detection. In general, when biological assays are developed, the biotin-streptavidin interaction is especially well suited to be measured considering that it forms a 9 ACS Paragon Plus Environment


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specific and strong bond. In our case, the AuBPs have been conjugated with the newly prepared p-ATP activated biotin by ligand exchange. The p-ATP@Biotin binds to the AuBP surface through the thiol group (-SH), representing not only the Raman-label but also the linkage to the protein (see Figure S1). The LSPR response of the p-ATP@Biotin-AuBPs before and after the specific streptavidin detection is presented in Figure 4A. As previously showed, the as-synthesized AuBPs exhibit in aqueous solution a weak transversal LSPR band at 512 nm and a strong longitudinal LSPR band at 793 nm (Figure 4A-black spectrum). When AuBPs are coupled with p-ATP@Biotin, a red-shift of 11 nm and 1 nm of the longitudinal and transversal LSPR, respectively, is observed compared to the extinction bands of AuBPs, due to the surface RI changes (Figure 4A-red spectrum). Additionally, the adsorption of p-ATP@Biotin onto the AuBPs surface is also proved by the sensitive red shift of the characteristic band of free Biotin in the UV region after the conjugation with the AuBPs (see Figure S6). Subsequently, the capture of the target protein by the biotinylated hybrid system leads to an additional 2 nm shift to the red of the longitudinal LSPR of p-ATP@Biotin -AuPBs (Figure 4A, green spectrum), confirming their specific detection and the high sensitivity of the bipyramids to their surface RI changes. In order to get a better insight into the experimental LSPR response of the AuBPs and their biosensing ability, we conducted numerical simulations by employing the Finite-Difference Time-Domain (FDTD) method (using the commercially available FDTD solutionsTM software from Lumerical Inc.36). For a detailed theoretical calculation please see the Supporting Information. The simulated optical response of AuBPs presented in inset of Figure 4A shows LSPR shifts of 11 nm and 2 nm after binding with p-ATP@Biotin and streptavidin, in good agreement with the experimentally obtained LSPR results. As pointed out in the Introduction section, Raman spectroscopy can be an ultrasensitive tool for quantitative detection and analysis of target analyte at low concentration, even in aqueous solution. The enhanced electromagnetic field (EM) arising at the surface of AuNPs, when the incident laser light is tuned to the LSPR band, highlights the unique Raman “fingerprint” of target analytes located in contact with metal or in its close proximity via SERS effect.37 For this reason, beyond LSPR spectroscopy, which allows the specific detection of streptavidin through the plasmonic red-shift due to the increase of the local refractive index, the biotin-streptavidin interactions are also demonstrated by SERS examination. In this context, we have performed comparative SERS measurements on p-ATP@Biotin-AuBPs before and after the specific targeting of streptavidin in solution (see Figure 4C). Firstly, the successful grafting of the p-ATP@Biotin onto the AuBPs surface is demonstrated by the observation of the p-ATP characteristic bands at 391 cm-1, 1079 cm-1, 1176 cm-1 and 1591 cm-1 35, together with the vibrational SERS bands associated with biotin structures at 746 cm-1 (CN2 wagging), 875 cm-1 (CH2 rocking), 970 cm-1 (C-N stretching -ureido ring), 1010, 1042 cm-1 (C-C stretching valeric acid chain), 1239 cm-1 (C-N stretching + N-H bending), 1285 cm-1 (CH2 wagging), 1456 cm-1 (CH2 rocking)38,39, correlating well with 10 ACS Paragon Plus Environment

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the vibrational bands obtained by DFT simulated spectra of p-ATP-Au (Figure 4C – spectrum a) and pATP@Biotin-Au (Figure 4C- spectrum b).

Figure 4. (A) Extinction spectra of the as-synthesized AuBPs (black), p-ATP@Biotin-AuBPs before (red) and after (green) the specific streptavidin detection. Insets show the close-up of the experimental (solid spectra) and FDTD simulated extinction spectra obtained for the considered AuBPs (dotted spectra); (B) Calculated │E/Eo2│ map for a AuBP with the length of 84 nm and diameter of 30 nm under laser excitation at 785, obtained using FDTD calculations; (C) DFT simulated Raman spectra of pATP@Au (spectrum a) and p-ATP@Biotin-Au, respectively (spectrum b) together with experimentally recorded SERS spectra of p-ATP@Biotin-AuBPs before (spectrum c) and after interaction with



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streptavidin (spectrum d). Final concentration of streptavidin is 10-6 M; (D) Assignment of the major SERS bands in the spectrum of biotin-streptavidin complex as illustrated in Fig. 4C.

After the specific binding with streptavidin, a change in the SERS fingerprint can be observed in Figure 4C-spectrum d, the major vibrational SERS bands being identified and assigned in Figure 4D. This strong interaction with the biotinylated plasmonic system is also evidenced by the appearance of characteristic Raman bands of the protein. In particular, a few new bands at 751 cm-1, 796 cm-1, 879 cm-1, 1010 cm-1 and 1349 cm-1 assigned to Tyr and Trp residues are observed, suggesting the successful recognition reaction. These spectroscopic observations are evidences of the biotin-streptavidin interaction alongside with the predominant presence of phenylalanine (Phe) at 1042 cm-1.39 Moreover, a change in the secondary structure of streptavidin upon specific interaction cannot be excluded considering the presence of the characteristic amide III bands around 1282 cm-1 -associated with α-helix structure and around 1224 cm-1 -corresponding to β-sheet structure.39,40 Next, the SERS performance of our designed AuBPs nanosensor was further investigated for quantitative analysis by incubation with varying concentrations of streptavidin target (see Supplementary Information). Furthermore, in order to understand the source of the electromagnetic enhancement in the SERS measurements, we also computed the electric field intensity distribution around a single AuBP using a 0.4 nm grid. Figure 4B illustrates the relative electric field intensity ((E/E0)2) calculated at the surface of an individual AuBP at the wavelength of 785 nm, according to the laser line employed in the SERS experiments. As a result, a large electromagnetic field at the two sharp AuBP’s tips can be observed when irradiating close to the longitudinal SPR band. This strong local EM field enhancement is responsible for the large increase of the experimentally recorded SERS signal. Finally, in order to evaluate the potential applicability of the designed nanosensor to real clinical diagnostics, the anti-human IgG -human IgG interaction was further chosen to be detected considering the vital role of IgG played in the immune system, especially for the determination of infectious and immunerelated diseases.20 In our case, the biosensing protocol is schematically illustrated in Figure 5A, presenting the steps involved in the fabrication of the dual nanosensor for the detection of human IgG. Figure 5B shows the monitorization of the wavelength shift of the LSPR response of AuBPs induced by the immunological reaction corresponding to different biofunctionalization steps involved in the fabrication of colloidal LSPR nanosensor. As a result, the grafting of the p-ATP Raman reporter (herein 10 mM) on the AuBPs surface resulted in a red-shift of 20 nm in the LSPR wavelength (Figure 5B red spectrum), which is closed to the theoretical LSPR shift value of 21.3 nm calculated from the equation 1. Subsequently, after the immobilization of the anti-human IgG on the functionalized AuBPs surface, we 12 ACS Paragon Plus Environment

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recorded another supplementary 16 nm red-shift of the AuBPs response (Figure 5A-blue spectrum) and a further red shift of 5 nm after the specific binding of human IgG to the anti-human IgG, indicating thus the successful formation of the immunocomplex and the feasibility of the AuBPs to operate as LSPR nanosensors.

Figure 5. (A) Schematic illustration representing the functionalization steps involved in the fabrication of the AuBPs immunosensor for human IgG detection; (B-C) Dual LSPR-SERS response of AuNBs before (black spectrum) and after their chemical modification with p-ATP (red spectrum), the anti-human IgG immobilization (blue spectrum) and the human IgG specific detection (orange spectrum). Additionally, the implementation of AuBPs as SERS-based immunoassays in solution was also proved in Figure 5C. Specifically, the detection of the recognition interactions was demonstrated by changes of the SERS fingerprint after the specific binding of the target human IgG (Figure 5C- orange spectrum) to the anti-human IgG (Figure 5C- blue spectrum). Concretely, in addition to the recorded characteristic SERS bands of p-ATP, a change appeared in the SERS intensity ratio of 1003 to 1033 cm-1 when target human IgG was adsorbed on the anti-human IgG@AuBPs. This change of the mentioned bands (i.e. 1003/1033), assigned to the symmetric C-C stretching and C-H in plan deformation vibrations of Phe residues, was 13 ACS Paragon Plus Environment


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also observed by Han et al.42, and was assigned to demonstrate the specific antigen – antibody interaction, the authors concluding the IgG-concentration-dependent variation function of the average intensity ratio of 1003/1033 cm-1 bands. Additionally, the amide III band at 1282 cm-1 associated with the α-helix structure is red-shifted to 1293 cm-1, caused probably by the conformational changes after the interaction with human IgG39. In general, the SERS spectra can be used to identify the target proteins considering that the recorded vibrational spectrum is directly related to the molecular structure, providing thus the unique signature of the protein. Although SERS is particularly informative as transducer for the structure and molecular orientation of the first layer of adsorbates41, herein we were able to detect the specific antihuman IgG – human IgG binding event which occurs slightly away from the first layer. Nevertheless, it is conceivable that such a binding event could induce conformation changes in the first layer of the recognition protein, located not far from the high electromagnetic field at AuBPs tips to be readout by SERS. Therefore, the protein-protein binding event was proved in our case by indirect SERS detection, as some of the SERS bands assigned to the recognition anti-human IgG are susceptible to transduce conformational changes once human IgG is linked and, consequently, making it possible to probe immunoassay interaction. All these spectral modifications clearly prove the detection of the human IgG using our developed AuBPs SERS immunosensor functionalized with anti-human IgG, confirming its capability to be further developed for point-of-care testing. CONCLUSIONS In this work, we focused on developing a new dual-modal LSPR/SERS nanoplatform based on gold bipyramidal-shaped nanoparticles in aqueous solution as a “proof-of-concept” immunosensor through the feasibility of detecting the specific biotin-streptavidin recognition interaction. Such plasmonic nanoplatforms take advantage of the unique optical properties of AuBPs, based on the spectral sensitivity of LSPR to the dielectric properties of the surrounding environment and the presence of extremely enhanced electromagnetic field at their tips which is able to improve target analyte detection via SERS. Firstly, successful grafting of p-ATP chemically labelled Biotin at the tips of the AuBPs, as recognition element for streptavidin detection, was proved by successive red shifts of the longitudinal LSPR associated with the increase of the local refractive index, proving the LSPR-based immunosensor capabilities of the designed nanoplatforms. Furthermore, considering that the molecular identification of the biotin-streptavidin interaction remains elusive by LSPR investigation alone, by exploiting the advantages of the SERS technique, we were able to identify the capture of the target protein to the biotinylated system by identifying the Raman bands of the amino acids involved into the recognition interaction. As such, SERS has been implemented herein in combination with LSPR-based sensing in immuno-analysis using AuBPs as optical transducers for the first time. Furthermore, the benefit of the 14 ACS Paragon Plus Environment

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ultrasensitive detection abilities of SERS technique, allows us to achieve a LOD of 10-12 M streptavidin concentration using our designed AuBPs nanoplatforms. Secondly, we demonstrated the capability of AuBPs to be implemented as dual LSPR-SERS immunosensors in solution for real clinical diagnostics by investigating the AuBP’s performance to transduce the specific anti-human IgG- human IgG binding event. These findings could be used as groundwork for the development of further immunoassay designs for real-time detection of relevant biomarkers in very low amount.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Materials, Gold bipyramids synthesis, and Characterization methods; Schematic illustration of the grafting of p-ATP@Biotin onto the AuBP surface; The assignment of the IR bands; TEM image of the selected AuBPs, their corresponding histogram and the Zeta Potential data of AuBPs 793; Bulk refractive index sensitivity; Surface refractive index sensitivity; Finite-Difference Time-Domain (FDTD) simulation; Calculation of limit of detection (LOD).

AUTHOR INFORMATION Corresponding Author *E-mail address: [email protected] (Monica Focsan); Tel: +40 264 454554/116; Fax: +40 264 591906 ORCID Ana Maria Craciun 0000-0002-6561-0972 Stephane Parola 0000-0001-7560-988X Simion Astilean: 0000-0002-9975-5651 Monica Focsan: 0000-0001-6735-5146 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by CNCS-UEFISCDI Romania, under the projects number PN-II-CT-ROFR2014-2-0049, PN-II-PT-PCCA-2013-4-1961 and PHC Brancusi 32656UE. 15 ACS Paragon Plus Environment


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Scheme 1. Schematic illustration of the biosensing protocol steps employed for the development of the plasmonic nanosensor. 144x18mm (300 x 300 DPI)

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Figure 1. (A) Schematic representation of the conjugation of NHS-Biotin with p-ATP in order to endow chemical affinity to the gold core; (B) DFT Simulation and experimental FT-IR spectra of p-ATP (spectra a and c) and p-ATP@Biotin (spectra b and d); (C) Assignment of the major IR bands in both simulated and experimental spectra of p-ATP and p-ATP@Biotin, respectively. 165x106mm (300 x 300 DPI)


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Figure 2. (A) Extinction spectra of the synthesized AuBPs with different aspect ratios ranging from 2.9 to 5.79; (B) Representative TEM image of the selected AuBPs with the longitudinal plasmon band located at 793 nm. 165x70mm (300 x 300 DPI)



Figure 3. (A) SERS spectra of p-ATP grafted on AuBPs surface (denoted as p-ATP@AuBPs) presented in Fig. 2A using 785 nm excitation laser. The dotted spectrum represents the simulated normal Raman spectrum of p-ATP; (B) Normalized extinction spectra of AuBPs before (solid spectra) and after (dotted spectra) the adsorption of p-ATP molecules. 165x69mm (300 x 300 DPI)


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Figure 4. (A) Extinction spectra of the as-synthesized AuBPs (black), p-ATP@Biotin-AuBPs before (red) and after (green) the specific streptavidin detection. Insets show the close-up of the experimental (solid spectra) and FDTD simulated extinction spectra obtained for the considered AuBPs (dotted spectra); (B) Calculated │E/Eo2│ map for a AuBP with the length of 84 nm and diameter of 30 nm under laser excitation at 785, obtained using FDTD calculations; (C) DFT simulated Raman spectra of p-ATP@Au (spectrum a) and pATP@Biotin-Au, respectively (spectrum b) together with experimentally recorded SERS spectra of pATP@Biotin-AuBPs before (spectrum c) and after interaction with streptavidin (spectrum d). Final concentration of streptavidin is 10-6 M; (D) Assignment of the major SERS bands in the spectrum of biotinstreptavidin complex as illustrated in Fig. 4C. 139x158mm (300 x 300 DPI)



Figure 5. (A) Schematic illustration representing the functionalization steps involved in the fabrication of the AuBPs immunosensor for human IgG detection; (B-C) Dual LSPR-SERS response of AuNBs before (black spectrum) and after their chemical modification with p-ATP (red spectrum), the anti-human IgG immobilization (blue spectrum) and the human IgG specific detection (orange spectrum). 165x106mm (300 x 300 DPI)


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Gold NanoBipyramids Performing as Highly ... - ACS Publications

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