Karim Aissou
email: karim.aissou@umontpellier.fr

Maximilien Coronas

Daniel Hermida-Merino

Eduardo Solano

Didier Cot

Stéphanie Roualdes

Denis Bouyer

Damien Quemener

Nanoporous Double-Gyroid Structure from ABC Triblock Terpolymer Thick Films

Keywords: Solvent vapor annealing, block copolymer membrane, self-assembly, doublegyroid, long-range order

The creation of nanostructured materials with a triply periodic minimal surface (TPMS), defined as a zero mean curvature surface having periodicity in three dimensional space, is an emerging solution to optimize transport (i.e., the ion-conductivity and hydraulic permeability) through the next-generation of electrolyte and ultrafiltration (UF) membranes.

Here, we used an amphiphilic ABC-type block copolymer (BCP) (namely, polystyrene-blockpoly(2-vinylpyridine)-block-poly(ethylene oxide) (PS-b-P2VP-b-PEO)) to generate symmetric thick films (8 µm) composed entirely of a TPMS-based structure, consisting of a PS matrix with a double gyroid (DG) minimal surface and hydrophilic stimuli-responsive (P2VP/PEO) nanochannels. To produce the core/shell DG-structured monoliths, we used a process combining the non-solvent induced phase separation (NIPS) process with a solvent vapor annealing (SVA) treatment. From such symmetric ABC-type BCP thick films generated by NIPS-SVA, a mean hydraulic permeability as high as 514 L h -1 m -2 bar -1 was measured. This mean value revealed to be nearly equal to that of asymmetric PS-b-P2VP-b-PEO membranes manufactured by NIPS, which have a substructure with an implicit irregular and random distribution of the internal pore structure and a skin layer with P2VP/PEO nanopores arranged into a hexagonal array.

Introduction

Improving transport in mechanically robust nanoporous membranes by creating effective pathways is very important for improving the heat dissipation [START_REF] El Sachat | Nanoscale Mapping of Thermal and Mechanical Properties of Bare and Metal-Covered Self-Assembled Block Copolymer Thin Films[END_REF] as well as the ionic conduction in fuel cells [START_REF] Kobayashi | Gyroid structured aqua-sheets with sub-nanometer thickness enabling 3D fast proton relay conduction[END_REF] and batteries [START_REF] Morris | Harnessing the Power of Plastics: Nanostructured Polymer Systems in Lithium-Ion Batteries[END_REF][START_REF] Shi | Nanostructured Conductive Polymers for Advanced Energy Storage[END_REF][START_REF] Yan | Gyroid and Other Ordered Morphologies in Single-Ion Conducting Polymers and Their Impact on Ion Conductivity[END_REF] or optimizing the figure of merit of separation-based materials for valuable ultrafiltration (UF) applications. [START_REF] Abetz | Isoporous Block Copolymer Membranes[END_REF][START_REF] Nunes | Block Copolymer Membranes for Aqueous Solution Applications[END_REF][START_REF] Zhang | Nanoporous Membranes Generated from Self-Assembled Block Polymer Precursors: Quo Vadis ?[END_REF][START_REF] Hampu | Next-generation ultrafiltration membranes enabled by block polymers[END_REF] However, controlling the orientation of porous nanochannels at the scale of membrane thickness (i.e., several micrometers) remains a major challenge to avoid the formation of undesirable dead ends that limit the transport properties.

An attractive way to optimize transport within the membrane without compromising its overall mechanical properties is to use porous materials with a triply periodic minimal surface (TPMS). [START_REF] Feng | Triply periodic minimal surface (TPMS) porous structures: from multi-scale design, precise additive manufacturing to multidisciplinary applications[END_REF] By definition, TPMS is a minimal surface with zero mean curvature that extends periodically in all three directions. These properties provide an excellent solution for the fabrication of advanced nanoporous membranes with controllable critical parameters (e.g., the connectivity of pathways and tortuosity) since TPMS-based structures can be precisely expressed by mathematical functions. [START_REF] Luo | Macroscopic transport properties of Gyroid structures based on pore-scale studies: permeability, diffusivity and thermal conductivity[END_REF] Among the different TPMS geometries, the doublegyroid (DG) is an intriguing bicontinuous phase that has recently gained interest in improving both ion transport in energy conversion and storage devices [START_REF] Crossland | A Bicontinuous Double Gyroid Hybrid Solar Cell[END_REF][START_REF] Choudhury | Nanoporous Cathodes for High-Energy Li-S Batteries from Gyroid Block Copolymer Templates[END_REF][START_REF] Werner | Block Copolymer Derived 3-D Interpenetrating Multifunctional Gyroidal Nanohybrids for Electrical Energy Storage[END_REF] and water transport through UF membranes. [START_REF] Park | Giant Gyroid and Templates from High-Molecular-Weight Block Copolymer Self-assembly[END_REF] The DG structure is formed by two mutually interwoven network channels, arranged in a cubic lattice with Ia3 ̅ d symmetry, where each channel is inherently interconnected in three dimensions via three-way junctions and the very smooth gyroid minimal surface separating the two networks ensures an excellent mechanical robustness of the material.

One of the most promising approaches to produce nanoporous materials with a DG structure is to use block copolymers (BCPs) as templates since BCPs are able to self-assemble into unique co-continuous nanostructures such as the cubic DG (space group 230) and double diamond (space group 224) phases and the orthorhombic Fddd (space group 70) morphology. [START_REF] Meuler | Ordered Network Mesostructures in Block Polymer Materials[END_REF][START_REF] Aissou | Periodic Bicontinuous Structures Formed on the Top Surface of Asymmetric Triblock Terpolymer Thick Films[END_REF] Following this strategy, water transport in several nanoporous materials derived from the self-assembly of BCPs into a DG structure has been examined in the literature. [START_REF] Park | Giant Gyroid and Templates from High-Molecular-Weight Block Copolymer Self-assembly[END_REF][START_REF] Li | Ultrafiltration by Gyroid Nanoporous Polymer Membranes[END_REF][START_REF] Li | Gyroid Nanoporous Membranes with Tunable Permeability[END_REF] However, a selective removal of the minor block has been systematically used to create the bicontinuous porosity within UF membranes, thereby leading to hydrophobic materials that can have a limited permeability and can be more easily subject to fouling if a post-modification treatment is not applied. [START_REF] Li | Ultrafiltration by Gyroid Nanoporous Polymer Membranes[END_REF] To overcome this issue, we herein propose to use an amphiphilic ABC-type BCP (namely, polystyrene-block-poly(2-vinyl pyridine)-block-poly(ethylene oxide) (PS-b-P2VP-b-PEO)) to generate permeable core/shell DG-structured monoliths with hydrophilic stimuli-responsive (P2VP/PEO) nanochannels. To achieve a reproducible DG structure, the PS-b-P2VP-b-PEO (S:V:EO  65:21:13, 69.5 kg.mol -1 ) films were blended with 30 wt. % of PS-b-PEO (S:EO  70:30, 39 kg.mol -1 ) chains while the DG-structured monoliths were generated by combining the non-solvent induced phase separation (NIPS) process with a solvent vapor annealing (SVA) treatment. Following this method, the NIPS-based terpolymer thick films, consisting of an asymmetric and highly porous sponge-like substructure topped by a dense thin layer with nanopores arranged in a hexagonal array, were exposed to a chloroform vapor to produce a well-developed DG structure within the entire film thickness. The mean hydraulic permeability value (514 L h -1 m -2 bar -1 ) of symmetric DG-structured materials, which was favorably interpreted using the Hagen-Poiseuille law, was also compared with the mean value (690 L h -1 m -2 bar -1 ) of their asymmetric homologues (i.e., of the same material) generated by the conventional NIPS method to evaluate the transport efficiency within the TPMS-based structure.

Results

The cross-sectional SEM views presented in Figure 1a terpolymer solution in a di-solvent mixture of 1,4-dioxane/tetrahydrofuran (DOX/THF: 80/20 by weight). The di-solvent mixture was subsequently evaporated for 60s at room temperature (RT) to form a dense air surface layer (see Fig. 1b). The blended PS-b-P2VP-b-PEO membrane was then immersed into a heptane bath at RT for 5 min to create a sponge-like substructure (see inset). Note that a dense bottom layer is also formed at the opposite interface of the PS-b-P2VPb-PEO/PS-b-PEO thick film (see Fig. 1c). Fortunately, this undesirable dense bottom layer did not affect the water permeability capabilities of the terpolymer membrane (i.e., permeability remains high, see the water flux results discussed hereafter). To know more about the morphology generated on the membrane top surface during the NIPS process that is not at the equilibrium, the atomic force microscopy (AFM) technique was The corresponding Delaunay triangulation shows that this trivial 2D array has a high density of defects ( = 0.3) such as dislocations and disclinations that delimit small grains (see Fig. 2b).

In addition, a mean pore diameter of 16.4 nm with a large standard deviation ( = 4.6 nm) was also measured (see Fig. S1b). These results confirm that stabilizing a long-range ordered nanostructure by using a NIPS process remains a major challenge. shows that a double-gyroid (DG) structure, exhibiting a typical double-wave pattern formed within large grains, is produced on the film top surface after the SVA treatment (5h, CHCl3).

Interestingly, the high magnification AFM topographic image focused on a single grain orientation reveals this 3D nanostructure is well-developed since the characteristic spots of the double wave pattern, consisting of small-and large-amplitude oscillations and corresponding to the (211) crystallographic plane of the DG structure, are clearly visible on its associated bidimensional fast Fourier transform (2D-FFT) (see Fig. 3b-c). Moreover, the low magnification (7 x 7 µm) AFM topographic image presented in Figure S2 suggests that a preferential orientation parallel to the air surface of the (211) crystallographic plane occurs which is fully supported by the grazing incidence small-angle X-ray scattering (GISAXS) data (see Fig. S3). Indeed, the GISAXS pattern corresponding to a solvent-annealed (5h, CHCl3) PS-b-P2VP-b-PEO/PS-b-PEO thick film well matches with the (121) and (220) Bragg diffraction spots as reported by the Ree group, [START_REF] Marques | Self-Assembly in Casting Solutions of Block Copolymer Membranes[END_REF][START_REF] Lee | Structural Analysis of Block Copolymer Thin Films with Grazing Incidence Small-Angle X-Ray Scattering[END_REF] confirming that a preferential {121} orientation occurs within the entire film thickness of the terpolymer material. From this GISAXS pattern, the unit cell of the DG structure (aG) was determined to be 75.8 nm, based on the diffracted (202 ̅ ) peak at 𝑞 𝑦,(202 ̅ ) ∼ 0.234 nm -1 (aG = (2π × 2√2)/ 𝑞 𝑦,(202 ̅ ) ), while the period of the (121) plane was estimated to be 30.9 nm. 4a-c). It is worth noting that the densification process occurring during the SVA treatment greatly reduces the thickness of blended terpolymer monoliths since a thickness of 8 µm was measured from SEM images. This behavior has been also observed in previous studies. [START_REF] Aissou | Periodic Bicontinuous Structures Formed on the Top Surface of Asymmetric Triblock Terpolymer Thick Films[END_REF][START_REF] Aissou | Bicontinuous Network Nanostructure with Tunable Thickness Formed on Asymmetric Triblock Terpolymer Thick Films[END_REF][START_REF] Aissou | Asymmetric Solvent-Annealed Triblock Terpolymer Thick Films Topped by a Hexagonal Perforated Lamellar Nanostructure[END_REF] Interestingly, the SEM image in Figure 4d shows that the DG structure on the bottom surface is not clearly visible compared to the well-developed double wave pattern produced on the top surface (see inset). To elucidate the formation of the DG structure during the SVA treatment, an early stage of annealing under a chloroform vapor was captured. The AFM topographic view presented in Figure S4a shows that the solvent-annealed (45min, CHCl3) PS-b-P2VP-b-PEO/PS-b-PEO thick film produced on the top surface has a long range ordered nanostructure exhibiting a 31 nm period wavy line pattern. This pattern differs both from the hexagonal phase generated by NIPS and the double wave pattern observed after a longer SVA duration. In addition, it can be seen from the top view SEM images that the blended triblock terpolymer thick film generated by NIPS-SVA (45min, CHCl3) consists of a sponge-like substructure topped by a sub-100nm thick layer with a "wavy line and dot" pattern (see Fig. S4b-c). The GISAXS data indicate this morphology generated by NIPS-SVA (45min, CHCl3) presents similarities with the DG structure, as the intensity cut along the horizontal qy direction, taken around the Yoneda band, displays peaks that overlap the gyroid diffraction peaks at qy = 0.113, 0.154, 0.177, 0.202, and 0.234 nm -1 (see Fig. S5). However, the wavy line and dot morphology, presenting epitaxial relations with the (211) DG plane (notably the same period), shows lower peak intensities than that arising from the well-developed 3D structure manufactured by NIPS-SVA (5h, CHCl3). This phenomenon is probably due to the fact that the solvent-annealed (45min, CHCl3) PS-b-P2VP-b-PEO/PS-b-PEO film has an asymmetric architecture with a thick sponge-like substructure (> 10 µm), which undoubtedly increases the background noise during the GISAXS measurement. The presence of a sponge-like substructure occupying most of the terpolymer film thickness could also affect the formation of the double pattern since it has been demonstrated that the small and large amplitude oscillations of the (211) DG plane are welldeveloped only when the layer thickness is well above the size of aG. [START_REF] Aissou | Templated Sub-100-nm-Thick Double-Gyroid Structure from Si-Containing Block Copolymer Thin Films[END_REF] In other words, a metastable wavy line and dot phase similar to the one produced in this work was already stabilized when the film thickness was lower (or in the same range) than the unit cell dimension.

Importantly, this metastable phase was also observed theoretically during the cylinder-to-DG transition using the self-consistent field theory (SCFT), which implies that the order-order transition between the hexagonal cylinder and DG structures proceeds epitaxially by a nucleation and growth mechanism. [START_REF] Matsen | Cylinder ↔ Gyroid Epitaxial Transitions in Complex Polymeric Liquids[END_REF] As the flow rate of water through symmetric membranes composed entirely of a welldefined DG structure has rarely been reported due to the complexity of manufacturing a such porous phase, the results obtained in this study are compared with those predicted using the conventional the Hagen-Poiseuille (H-P) fluid flow theory defined as follows:

𝐽 = 𝑁 𝜋 𝑑 4 ∆𝑃 128 × 𝜇 × 𝜏 × 𝐿
where J is the flux, N is the areal density of pores, d is the pore diameter, P is the transmembrane pressure,  is the viscosity of water (1  10 -3 Pa.s at 20°C),  is the pore tortuosity ( = 1.371 -0.250 × (1 - 𝑃𝑆 ) + 0.086 × (1 - 𝑃𝑆 ) 2 = 1.295 for the gyroid structure) [START_REF] Luo | Macroscopic transport properties of Gyroid structures based on pore-scale studies: permeability, diffusivity and thermal conductivity[END_REF] , and L is the nanochannel length (approximated here by the film thickness  8 µm).

If we assume that the blended terpolymer monolith has a PS volume fraction,  𝑃𝑆 , of 0.66, a pore radius, Rpore, of 17.7 nm (2𝜋𝑅 𝑝𝑜𝑟𝑒 2 / 𝑎 𝐺 2 =(1 - 𝑃𝑆 )), and thus an areal density, N, of 3.46  10 14 pores/m² (N𝜋𝑅 𝑝𝑜𝑟𝑒 2 = (1 - 𝑃𝑆 )) can be determined by approximating the DG morphology to two tortuous and unconnected porous nanochannels per unit cell (see Fig. 5a).

Using these parameter values, a theoretical permeability, J/ΔP, of 463 L m -2 h -1 bar -1 can be calculated, which agrees quite well with the mean water permeance value (514 L h -1 m -2 bar -1 ) measured from three different solvent-annealed (5h, CHCl3) terpolymer films deposited on porous (0.1 µm) hydrophilic PVDF supports (see Fig. 5b). Note that the mean water permeance value corresponds to the slope of the linear fits based on the Darcy's law, and the value of the coefficient of determination, R², close to unity (R²  0.99) indicates that the DG-structured monoliths exhibit excellent stability to material failure even at 2 bar (water conditions: pH = 7 and T = 20°C). Conversely, the mean water permeance value (690 L h -1 m -2 bar -1 , R²  0.99) of the asymmetric terpolymer films produced by NIPS deviates strongly from the theoretical value calculated using the H-P law, indicating that the sponge-like substructure contributes strongly to the permeability (in this study of 84%). [START_REF] Dorin | Designing Block Copolymer Architectures for Targeted Membrane Performance[END_REF] Indeed, an asymmetric membrane architecture comprising an ideal sponge-like substructure with no resistance to water transport and a 100 nm thick top surface with porous domains packed in a 40 nm period hexagonal lattice (N = 6.56  10 14 pores/m²) would reach a theoretical value of 4192 L m -2 h -1 bar -1 . In contrast to asymmetric terpolymer membranes generated by NIPS, the results show that the permeability value of the DG-structured monoliths is predictable. Moreover, this average value is also almost the same as the value measured from NIPS-based membranes since the error bars overlap. To extend the comparison, we recently studied the permeability of blended PS-b-P2VPb-PEO symmetric membranes composed entirely of a hexagonal perforated lamellar (HPL) phase. [START_REF] Aissou | Asymmetric Solvent-Annealed Triblock Terpolymer Thick Films Topped by a Hexagonal Perforated Lamellar Nanostructure[END_REF] These 9 µm thick PS-b-P2VP-b-PEO monoliths blended with 20 wt. % of short PS chains showed a water permeance of 30 L h -1 m -2 bar -1 , which is an order of magnitude lower than the mean value obtained for the DG-structured monoliths studied in this work. This result confirms that the HPL phase having PS perforations is not an optimized 3D-morphology to generate monoliths with efficiently interconnected porous nanochannels that are able to limit the tortuosity of the porous structure and obviate the need for domain alignment as the morphological excellence of the DG structure.

Conclusion

In summary, symmetric terpolymer monoliths composed entirely of a well-developed DG nanostructure were fabricated using a NIPS-SVA method. A mean average hydraulic permeance of 514 L h -1 m -2 bar -1 was measured for these DG-structured monoliths, showing a linear variation between J and the pressure difference. By approximating the DG morphology to two tortuous and unconnected porous nanochannels per unit cell, we found that the mean hydraulic permeability value of such symmetric terpolymer monoliths agrees well with the theoretical value calculated using the Hagen-Poiseuille law. This permeability value was found to be an order of magnitude higher than that measured from analogous PS-b-P2VP-b-PEO membranes composed entirely of a perforated hexagonal lamellar phase (HPL). Lastly, the use of PEO-based BCP chains to create bicontinuous porosity in UF membranes is highly desirable to produce nanopores with improved fouling resistance properties.

Experimental

GISAXS characterizations:

GISAXS experiments were performed on the Dutch-Belgian Beamline (DUBBLE) at the European Synchrotron Radiation Facility (ESRF) station BM26B in Grenoble. [START_REF] Portale | Polymer research and synchrotron radiation perspectives[END_REF] A monochromatic beam of 12 keV was set using a Si (111) double crystal monochromator. The sample (typical size of 150 mm 2 ) was shone with the X-ray beam with an incidence angle of 0.2º, that was above the critical angle of the polymer, ensuring full penetration of the X-ray beam in the material and hence, analyzing the full volume of the sample. [START_REF] Saito | Depth-Resolved Structure Analysis of Cylindrical Microdomain in Block Copolymer Thin Film by Grazing-Incidence Small-Angle X-ray Scattering Utilizing Low-Energy X-rays[END_REF] The 2D scattering patterns were collected with a PILATUS3 S 1M detector. The scattering vector and the sample to detector distance were calibrated using silver behenate as standard, obtaining a sample-to-detector distance of 7500 mm. [START_REF] Aissou | Asymmetric Solvent-Annealed Triblock Terpolymer Thick Films Topped by a Hexagonal Perforated Lamellar Nanostructure[END_REF] at pressures between 0.5 and 2 bar. The mass of water passing through the stacked materials (permeate) was recorded using a connected balance at regular time intervals for 10 min. Water temperature was maintained at 20°C during the measurements while the error bars were calculated from 3 different samples. In order to avoid a non-linear behavior of the water flux with the increase in pressure drop, a transmembrane pressure of 2.5 bar was systematically applied during 10 min on each terpolymer film prior measurements to determine their permeability performance.

  show a 14.5 µm thick PS-b-P2VPb-PEO/PS-b-PEO film blended with 30 wt. % of PS-b-PEO chains, which adopts an asymmetric architecture after the NIPS treatment. To generate this asymmetric and highly porous architecture, a square-shaped PS-b-P2VP-b-PEO/PS-b-PEO film was first drawn onto a (3 x 3 cm) silicon substrate by using a tape casting technique with 250µm gap from a 18 % wt.

Figure 1 :

 1 Figure 1: Cross-sectional SEM views of a PS-b-P2VP-b-PEO/PS-b-PEO thick film generated by NIPS showing (a) the entire material with a thickness of 14.5 µm and the presence of a graded sponge-like substructure with small and large pores near the (b) top and (c) bottom layer, respectively. Inset: magnified SEM image of the microporous (sponge-like) substructure formed within the asymmetric terpolymer thick film. Scale bars: 500 nm.

  used. The representative (4 x 4 µm) AFM topographic view indicates that the top surface of PSb-P2VP-b-PEO/PS-b-PEO thick films generated by NIPS is quite rough (see Fig. S1a) while the magnified AFM topographic image presented in Figure 2a shows the porous P2VP/PEO (black) nanodomains are arranged into a 2D hexagonal structure with a period of 40.1 nm.

20

 20

Figure 2 :

 2 Figure 2: (a) AFM topographic image and (b) its associated Delaunay showing the top surface of a NIPS-made PS-b-P2VP-b-PEO/PS-b-PEO thick film. The dark (P2VP/PEO) pores are arranged into a short-range ordered hexagonal array with a period of 40.1 nm. In the Delaunay triangulation, the small dots are six-fold coordinated pores, while the black and white big dots, indicating defect sites, are five-and seven-fold coordinated (P2VP/PEO) domains, respectively. The colormap shows the orientations of small grains (-30°-30°). Scale bars: 250 nm.

Figure 3 :

 3 Figure 3: (a) Topographic AFM image of a NIPS-SVA made (5h, CHCl3) PS-b-P2VP-b-PEO/PS-b-PEO thick film showing large grains formed by the DG structure on the material top surface. (b) Magnified AFM topographic view and (c) its 2D-FFT revealing the double wave pattern is well developed within its grain as the 2D-FFT exhibits sharp spots. Scale bars: 250 nm.

  To gain insight into the DG structure formed inside the PS-b-P2VP-b-PEO/PS-b-PEO thick film generated by NIPS-SVA, cross-sectional SEM images showing different regions of the terpolymer material were acquired. The cross-sectional images shown in Figure 4 indicate that the solvent-annealed (5h, CHCl3) PS-b-P2VP-b-PEO/PS-b-PEO thick film has lost its asymmetric architecture as well as its high porosity generated by NIPS (see Fig. 1). Indeed, the blended terpolymer material strongly densified during the SVA treatment to form a symmetric film entirely composed of the DG structure as shown on the cross-sectional SEM views taken close to the top and bottom surfaces and in the middle of the blended PS-b-P2VP-b-PEO monolith (see Fig.

Figure 4 :

 4 Figure 4: Cross-sectional SEM views of a PS-b-P2VP-b-PEO/PS-b-PEO monolith generated by NIPS-SVA (5h, CHCl3) showing the DG structure in the vicinity of the (a) top, (b) middle and (c) bottom film regions. (d) Top view SEM image revealing the nanoporous morphology formed on the bottom surface. Inset: top view SEM image showing the double wave pattern produced on the membrane top surface and the DG structure formed within the blended terpolymer film. Scale bars: 500 nm.

Figure 5 :

 5 Figure 5: (a) (left) Schematic representation of the DG unit cell (a cube with sides equal to aG) showing only the (red and blue) inter-material dividing surfaces (IMDSs) separating the two interpenetrating three-dimensional continuous networks from the matrix. (right) To apply the H-P law, each network made of interconnected tubes was simplified to a tortuous nanochannel having a circular cross-section. (b) Water fluxes, J, of the NIPS made PS-b-P2VP-b-PEO/PSb-PEO materials exposed to a CHCl3 vapor for different times: (red dots) 0 min and (blue dots) 5h. The error bars are the standard deviations of the mean relative flux values calculated for 3 different samples.

Material: 1 , 4 -

 14 Dioxane (DOX, 99.5 %), tetrahydrofuran (THF, 99%), chloroform (CHCl3, 99%), poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS, 2.8% wt. in water) and PVDF membranes (Durapore, 0.1 µm and 47 mm) were purchased from Sigma Aldrich. Silicon substrates were provided from Silicon Materials (Si-Mat). The PS-b-P2VP-b-PEO (S:V:EO  65:21:13, 69.5 kg.mol -1 ) and PS-b-PEO (S:EO  70:30, 39 kg.mol -1 ) BCP chains, used in this work, were purchased from Polymer Source inc (Canada).Asymmetric PS-b-P2VP-b-PEO/PS-b-PEO thick films generated by NIPS:Asymmetric PS-b-P2VP-b-PEO films were manufactured by NIPS as follows: A 18% wt.terpolymer solution in a di-solvent mixture (DOX/THF: 80/20 by weight) was first prepared and heated for 30 minutes at 70°C in order to achieve the dissolution of the terpolymer. The terpolymer solution was cooled down at RT before any uses. To produce the PS-b-P2VP-b-PEO/PS-b-PEO films, 120 µL of the terpolymer solution was dropped onto (3 x 3 cm) silicon substrates and was sprayed homogeneously by using a tape casting technique with a 250µm blade. To fabricate asymmetric PS-b-P2VP-b-PEO/PS-b-PEO films topped by a 2D hexagonal nanoporous structure, the previously casted solution was kept in the room during 60s at RT to allow the solvents to evaporate, and then the terpolymer materials were immersed into a (non solvent) heptane bath at RT for 5 min to produce a sponge-like substructure.DG-structured PS-b-P2VP-b-PEO/PS-b-PEO monoliths generated by SVA:To achieve monoliths entirely composed of a DG structure, the terpolymer films manufactured by NIPS were exposed to a CHCl3 vapor for 5h in order to promote the mobility of BCP chains until reaching the formation of the DG structure. Here, a continuous flow system was used to control the CHCl3 vapor pressure (32 sccm CHCl3 + 8 sccm N2) in the SVA chamber as described previously.[START_REF] Gotrik | Morphology Control in Block Copolymer Films Using Mixed Solvent Vapors[END_REF] Note that the temperature of the CHCl3 tank was kept at 20°C during the SVA process. AFM and SEM imaging: Atomic force microscopy (AFM Nano-Observer, CSInstruments) was used in tapping mode to characterize the surface morphology of PS-b-P2VP-b-PEO/PS-b-PEO films. Silicon cantilevers (PPP-NCH, Nanosensors) with a typical tip radius of 5 nm were used. The resonance frequency of cantilevers was 235 kHz. Scanning electron microscopy (SEM, Hitachi S-4800) was used at an accelerating voltage of 5 kV to acquire top view and cross-section images of both asymmetric thick films and monoliths.

  Water flux performances: To perform the water flux tests, the blended PS-b-P2VP-b-PEO/PS-b-PEO films generated by NIPS-SVA (0h and 5h) were deposited on a sacrificial (water soluble) 100 nm thick PEDOT:PSS layer in order to facilitate the removal of the terpolymer layer from the substrate. For that purpose, silicon pieces were treated by an oxygen plasma in a home-made chamber (plasma conditions: 45 W, 75 mTorr O2, 10 min) prior the deposition of the sacrificial PEDOT:PSS layer (spin-coat conditions: speed = 1000 rpm, acceleration = 500 rpm.s -1 , time = 90s). The water permeability of the different PS-b-P2VP-b-PEO/PS-b-PEO films was measured in a 10 mL filtration cell (Amicon 8010 stirred cell) connected to a water reservoir and a compressed air line. The measurements were performed on 2.5 cm diameter PS-b-P2VP-b-PEO/PS-b-PEO material discs supported by a high permeable hydrophilic PVDF material

Acknowledgements

This work was performed within the support of the ANR JCJC AFM_Ring project, grant ANR-18-CE09-00xx of the French Agence Nationale de la Recherche.

Data Availability

Data supporting this research article are available from the corresponding author or first author on reasonable request.


Vincent Pellas

Fadoua Sallem

Juliette Blanchard

Antoine Miche

Sara Martinez Concheso

Christophe Méthivier

Michèle Salmain

Souhir Boujday

Silica-coated gold nanorods biofunctionalization for localized surface plasmon resonance (LSPR) biosensing

Keywords: Gold Nanorods, Silica Coating, Surface Chemistry, C -Shell, LSPR, Immunosensing

come

Introduction

Refractometric biosensors are analytical devices tightly associating a bioreceptor and an optical transducer whose response arises from the change of refractive index that occurs upon binding of the target to the bioreceptor at the solid/ liquid interface. Aside from the highly popular label-free surface plasmon resonance biosensors, localized surface plasmon resonance (LSPR) biosensors are gaining increasing interest because (1) they are cheaper label-free transducers, (2) they enable real time sensing, (3) they require less technically demanding instrumentation as compared to SPR and finally (4) they are easy to miniaturize [START_REF] Estevez | Trends and challenges of refractometric nanoplasmonic biosensors: A review[END_REF][START_REF] Sepúlveda | LSPR-based nanobiosensors[END_REF][START_REF] Soler | Label-free plasmonic biosensors for point-of-care diagnostics: a review[END_REF].

The LSPR phenomenon results from the interaction of light with metal nanostructures (typically coinage metal nanoparticles) that gives rise to characteristic extinction whose position depends on the nanostructure but typically ranges from the near-UV to the nearinfrared spectra range. The principle of LSPR-based biosensors relies on the shift of the LSPR band position of plasmonic nanostructures that happens when the refractive index (RI) of the surrounding medium changes. The change of local refractive index typically occurs when an antigen binds to antibody-coated gold nanoparticles that translates into a red shift of the plasmon band. Plasmonic nanostructures are characterized by the RI sensitivity factor that is strongly dependent on their size, shape and composition [START_REF] Chen | Gold nanorods and their plasmonic properties[END_REF]. Furthermore, the shift of the plasmon band position with respect to RI change is highly distance-dependent [START_REF] Nusz | Rational Selection of Gold Nanorod Geometry for Label-Free Plasmonic Biosensors[END_REF] and comparatively to SPR, the probing distance of LSPR is much shorter, that is comparable to biomacromolecules size, meaning that the bioreceptortarget complexes occupy the entirety of the evanescent field [START_REF] Tian | Gold Nanorods as Plasmonic Nanotransducers: Distance-Dependent Refractive Index Sensitivity[END_REF].

Among the variety of plasmonic nanoparticles synthetically accessible by bottom-up methods, gold nanorods (AuNR) are particularly well suited to design LSPR nanobiosensors [START_REF] Mayer | Localized Surface Plasmon Resonance Sensors[END_REF]. Besides the transverse plasmon band (t-LSPR) at ca. 520 nm, AuNR manifest a second extinction peak at higher wavelength known as the longitudinal plasmon band (l-LSPR) [START_REF] Murphy | Anisotropic Metal Nanoparticles: Synthesis, Assembly, and Optical Applications[END_REF]. The position of this band can be tuned from the visible to the near-IR spectral range according to the AuNR aspect ratio, i.e. the length divided by the width [START_REF] Chang | Mini Gold Nanorods with Tunable Plasmonic Peaks beyond 1000 nm[END_REF]. Most importantly, the longitudinal band of AuNR generally displays a high RI sensitivity [START_REF] Chen | Sensing capability of the localized surface plasmon resonance of gold nanorods[END_REF][START_REF] Pellas | Gold Nanorods for LSPR Biosensing: Synthesis, Coating by Silica, and Bioanalytical Applications[END_REF]. For instance, AuNR-coated glass chips exhibit a bulk RI sensitivity of 252 nm/RIU (Refractive Index Unit) that is 4 times that of nanospheres [START_REF] Marinakos | Plasmonic Detection of a Model Analyte in Serum by a Gold Nanorod Sensor[END_REF]. Consequently, binding events may be more sensitively detected with AuNR-based LSPR biosensors.

Few examples of AuNR-based LSPR biosensors operating in the solid phase with nanostructured flat substrates [START_REF] Mayer | A Label-Free Immunoassay Based Upon Localized Surface Plasmon Resonance of Gold Nanorods[END_REF] or in solution [START_REF] Yu | Multiplex Biosensor Using Gold Nanorods[END_REF][START_REF] Guo | Fabrication of Antihuman Cardiac Troponin I Immunogold Nanorods for Sensing Acute Myocardial Damage[END_REF][START_REF] Wang | Biorecognition-Driven Self-Assembly of Gold Nanorods: A Rapid and Sensitive Approach toward Antibody Sensing[END_REF][START_REF] Wang | Gold Nanorod Probes for the Detection of Multiple Pathogens[END_REF] have been reported to date. Moreover, the latter ones were mostly based on nanoparticle aggregation upon target binding, resulting in a large decrease of the LSPR band intensity. This limitation in the use of AuNRs for biosensing, despite their outstanding properties, is caused by the presence of the surfactant cetyltrimethylammonium bromide (CTAB)mandatory for their synthesis -which forms a strongly-bounded and positively-charged bilayer at their surface and significantly complicates their functionalization. In addition, CTAB can also induce non-specific binding of molecules via electrostatic interactions [START_REF] Pellas | Gold Nanorods for LSPR Biosensing: Synthesis, Coating by Silica, and Bioanalytical Applications[END_REF]. Grafting of PEG has been proposed to functionalize AuNR with bioligands [START_REF] Centi | Ready-to-use protein G-conjugated gold nanorods for biosensing and biomedical applications[END_REF]. Alternatively, coating of AuNR with a shell of silica screens the CTAB bilayer and enables further biofunctionalization via organosilane chemistry [START_REF] Lai | A multifunctional organosilica cross-linker for the bioconjugation of gold nanorods[END_REF]. Furthermore, mesoporous silica-coated AuNR (AuNR@SiO 2 ) maintain a high sensitivity to RI change [START_REF] Wu | Stable and Functionable Mesoporous Silica-Coated Gold Nanorods as Sensitive Localized Surface Plasmon Resonance (LSPR) Nanosensors[END_REF]. We, and others, have proved that the sensitivity factor could even be enhanced for AuNR@SiO 2 with respect to CTAB-coated AuNR, provided the silica shell thickness is thin enough (2 -4 nm) [START_REF] Song | Amplifying the signal of localized surface plasmon resonance sensing for the sensitive detection of Escherichia coli O157:H7[END_REF][START_REF] Pellas | Gold Nanorod Coating with Silica Shells Having Controlled Thickness and Oriented Porosity: Tailoring the Shells for Biosensing[END_REF]. Nevertheless, very few examples of AuNR@SiO 2 -based LSPR biosensors have been reported in the literature as their surface chemistry is still to be mastered [START_REF] Song | Amplifying the signal of localized surface plasmon resonance sensing for the sensitive detection of Escherichia coli O157:H7[END_REF][START_REF] Wang | Synthesis, Assembly, and Biofunctionalization of Silica-Coated Gold Nanorods for Colorimetric Biosensing[END_REF].

We have recently introduced homogeneous, solution-phase LSPR biosensors based on colloidal gold and core-shell Au@Ag nanospheres conjugated to an antibody for the detection of a bacterial toxin target [START_REF] Ben Haddada | Gold nanoparticle-based localized surface plasmon immunosensor for staphylococcal enterotoxin A (SEA) detection[END_REF][START_REF] Loiseau | Core-Shell Gold/Silver Nanoparticles for Localized Surface Plasmon Resonance-Based Naked-Eye Toxin Biosensing[END_REF]. We have also designed an efficient and reproducible procedure to cover AuNR with a ultrathin layer of silica and found that their RI sensitivity factor was enhanced by 30 % compared to CTAB-coated AuNR, which let us anticipate better analytical performances [START_REF] Pellas | Gold Nanorod Coating with Silica Shells Having Controlled Thickness and Oriented Porosity: Tailoring the Shells for Biosensing[END_REF]. More generally, silica surface functionalization is easily amenable via silane condensation on silanol groups [START_REF] Aissaoui | Silane Layers on Silicon Surfaces: Mechanism of Interaction, Stability, and Influence on Protein Adsorption[END_REF][START_REF] Boujday | Amorphous Silica as a Versatile Supermolecular Ligand for NiII Amine Complexes: Toward Interfacial Molecular Recognition[END_REF].

Herein we report the biofunctionalization of AuNR@SiO 2 via three different routes to design and test a label-free LSPR immunosensor of rabbit IgG operating in solution. This target is a convenient model to set up biosensors, enabling our own nanoprobe performances to be compared with the early designed nanoprobe reported by Ma et al. for the detection of human IgG [START_REF] Wang | Biorecognition-Driven Self-Assembly of Gold Nanorods: A Rapid and Sensitive Approach toward Antibody Sensing[END_REF]. In the first route, we took advantage of the negatively charged external silica shell to immobilize anti-rabbit IgG antibody by electrostatic physisorption. In the second and third route, the silica surface was reacted with thiol or aldehyde terminated silane, subsequently utilized to covalently attach anti-rabbit IgG antibody to the surface. The different steps of surface modification were deeply characterized and the biofunctionalization investigated in terms of both kinetics and thermodynamics. Finally, the resulting nanoprobes were applied to the detection of the rabbit IgG analyte and their analytical performances assessed and comparatively discussed.

Materials and methods

Chemicals and Reagents

Tetraethyl orthosilicate (≥99.0%, TEOS), cetyltrimethylammonium bromide (BioUltra ≥99.0%, CTAB), (3-mercaptopropyl)trimethoxysilane (≥95%, MPTMS), 5,5′-dithiobis(2nitrobenzoic acid) (≥98%; DTNB), tris(2-carboxyethyl) phosphine hydrochloride (≥ 98%, TCEP), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (≥ 80%, Sulfo-MBS), sodium cyanoborohydride (≥ 95%), fluorescein 5-isothiocyanate (≥ 90% isomer I, FITC), ammonium chloride (≥95.5%), bovine serum albumin (≥98%, BSA), rabbit IgG (≥95%), sodium dihydrogen phosphate (≥99.0%), and anhydrous disodium hydrogen phosphate (>99.0%) were purchased from Sigma-Aldrich. Hydrochloric acid (37%; HCl) was purchased from VWR chemicals. 3-Triethoxysilylbutyraldehyde (≥90%, TEBA) was purchased from Gelest. Glycerol (99.5%), sodium carbonate (≥99.8%), and sodium hydrogen carbonate (≥99.5%) were purchased from Humeau. Affinity-purified polyclonal goat anti-rabbit IgG (anti-rIgG) was purchased from Jackson Immunoresearch. All aqueous solutions were prepared with p p w (18.2 Ω.cm -1 ).

Characterization techniques

UV-Vis Spectroscopy. UV-Visible (UV-Vis) extinction spectra were recorded on a Cary 50 spectrophotometer (Varian, Inc.) and the analysis of colloidal dispersions was performed in the 400-1100 nm range using 1-cm optical pathlength plastic cuvettes. According to refs. [START_REF] Hendel | In Situ Determination of Colloidal Gold Concentrations with UV-Vis Spectroscopy: Limitations and Perspectives[END_REF][START_REF] Scarabelli | Tuning Gold Nanorod Synthesis through Prereduction with Salicylic Acid[END_REF], Abs 400 linearly correlates with the Au 0 concentration and Abs 400 = 1.2 at 400 nm corresponds to 100 % reduction of a 0.5 mM gold salt solution. According to this relation, the reduction yield of Au 3+ was estimated. Mathematical determination of λ max was done from the first order derivative spectra as recommended in refs [START_REF] Chen | Curvature of the Localized Surface Plasmon Resonance Peak[END_REF][START_REF] Chen | Inflection Point of the Localized Surface Plasmon Resonance Peak: A General Method to Improve the Sensitivity[END_REF].

Dynamic light scattering (DLS) and electrophoretic light scattering (ELS). DLS and ELS measurements were performed using a Litesizer™ 500 pp (A ) q pp w a 658 nm laser operating at 40 mW. The scattered light collection angle was set to 90°. The z p w Ωshaped capillary tube cuvette with an applied potential of 150V. Gold nanorods being anisotropic particles, their diffusion is associated to two characteristic times that correspond to two motions: rotational and translational. These two times lead to two signals in the particle size distribution, one of low intensity, at relatively low dimensions (ca. 10 nm equivalent hydrodynamic diameter) and associate to rotational diffusion, and the other, more intense, at larger dimensions (ca. 100 nm equivalent hydrodynamic diameter), associated to translational diffusion [START_REF] Liu | Dynamic light scattering for gold nanorod size characterization and study of nanorod-protein interactions[END_REF].

Transmission electron microscopy (TEM). TEM images of AuNR and AuNR@SiO 2 were obtained using a JEOL JEM 1011 transmission electron microscope (TEM) operating at an accelerating voltage of 100 kV. Samples were prepared by depositing a drop of water-diluted (x20) particle suspension onto a carbon-coated copper grid and drying the grid at room temperature before imaging. Size distribution of AuNR and silica shell thickness of AuNR@SiO 2 were established by counting a minimum of 200 particles and 200 shells using Image J software.

X-ray photoelectron spectroscopy (XPS).

XPS analyses were performed using a Scienta Omicron Argus X-ray photoelectron spectrometer q pp w A Kα radiation source (1486.6 eV) and operating at 280 W power. Analysis was performed under an ultrahigh vacuum (<10 -9 mbar). The photoelectrons were analyzed in a direction making an angle of 45° to the surface. Spectra were recorded at a passing energy of 100 eV for the survey spectrum and 20 eV for the S 2p region. Data analysis was carried out using Casa XPS v.2.3.15 software (Casa Software Ltd., U.K.). Colloidal suspensions (1.5 mL) were y g (10 9000 p ) 30 μ . T g p w p 10μ p 2 y .

Surface functionalization of core-shell gold-silica nanorods

Synthesis of gold nanorods (AuNR) and core-shell gold nanorods-silica (AuNR@SiO 2 ) AuNR were synthesized and characterized by TEM, UV-Vis spectroscopy, DLS and ELS following the protocol detailed in a previous work [START_REF] Pellas | Gold Nanorod Coating with Silica Shells Having Controlled Thickness and Oriented Porosity: Tailoring the Shells for Biosensing[END_REF]. The size of the synthesized AuNR is L= 102 (± 7) nm, l= 40 (± 2) nm with aspect ratio (AR) of 2.58 (± 0.2) and  max = 716 nm. A 3-nm silica layer was grown on gold nanorods as previously described [START_REF] Pellas | Gold Nanorod Coating with Silica Shells Having Controlled Thickness and Oriented Porosity: Tailoring the Shells for Biosensing[END_REF]. Briefly, prior to SiO 2 coating, the AuNRs suspension was diluted with water to adjust the Au 0 and CTAB concentrations to 1.6 and ca. 6 mM, respectively (CTAB concentration value is estimated based on the dilution of the fraction of growth solution remaining with the AuNRs after centrifugation). The initial pH was adjusted to 4.0 by adding HCl or NaOH solution (0.1 M) and kept under stirring for 20 min at 400 rpm. Then, 41 μ T acting as primer (100 mM) in EtOH were added under stirring (400 rpm) for 3 h. Next, 62 µL of TEOS in MeOH (20%) was added for a final Au/TEOS mole ratio of 1:1.80, and the suspension was stirred for 1 h at 800 rpm. The pH was adjusted to 8.0 using 0.1 M NaOH and the suspension was stirred for 20 min at 400 rpm before being kept without stirring for 20 h at room temperature. The resulting core-shell particles, referred to AuNR@SiO 2 , were sonicated and centrifuged at 7060 rcf for 15 min. The sedimented particles were washed four times with EtOH and twice with water and finally redispersed in 5 mL of water.

Silanes assembly on AuNR@SiO 2

AuNR@SiO 2 suspension (3.0 mL; surface area: 8.11x10 15 nm 2 , [AuNR@SiO 2 ] = 15.7 nM, OD max ~ 6.0) in EtOH: H 2 O (95:5 v/v) was sonicated for 5 min and then heated at 60°C under stirring for 20 min in the oven in order to homogenize the solution. Then, 1.25 µL of pure MPTMS (or 1.64 µL of TEBA) (corresponding to 100 eq/surface OH; 2.24 mM final) was added to the solution, which was stirred for 17 h at 60°C and 500 rpm. Particles were washed twice with ethanol and twice with Milli Q water by centrifugation (15 min, 9000 rpm) and stored in 3.0 mL of Milli Q water.

Quantification of thiol functions on AuNR@SiO 2 -MPTMS

Quantification of thiol groups in AuNR@SiO 2 -MPTMS was carried out by Ellman's reagent (DTNB) assay in triplicate [START_REF] Al Mahrooqi | Controlling the Size of Thiolated Organosilica Nanoparticles[END_REF]. AuNR@SiO 2 -MPTMS suspension (250 µL, 157 nM in 50 mM phosphate buffer pH 8) was mixed with DTNB solution (250 μ 50 µM in 50 mM phosphate buffer pH 8), incubated in the dark for 90 min and centrifuged for 10 min at 9,000 rpm. The concentration of released TNB 2-in the supernatant was determined from a calibration curve obtained from standard solutions of cysteine between 5 and 200 µM, giving  nm = 13450 M -1 .cm -1 .

Engineering solution-phase LSPR nanobiosensors

Bioconjugation of antibodies to AuNR@SiO 2 and AuNR@SiO 2 -MPTMS/TEBA Physisorption to AuNR@SiO 2 : Goat anti-rIgG (15 µL, 1.2 mg/mL) was added to a suspension of AuNR@SiO 2 [AuNR@SiO 2 ]=15.7 pM, OD max ~ 0.6, ~ 1.5 mL in 5 mM phosphate buffer pH 7.4). The mixture was stirred for 90 min at room temperature and solid BSA was added to a final concentration of 0.5% (w/v) to block the non-specific adsorption sites and the suspension was kept under magnetic stirring for 45 min before the final washing step (see below).

Chemisorption to AuNR@SiO 2 -MPTMS: TCEP solution (10 µL, 150 mM) was added to AuNR@SiO 2 -MPTMS suspension ([AuNR@SiO 2 ] = 15.7 pM, OD max ~ 0.6, ~ 1.5 mL in 5 mM phosphate pH 7.4) in order to reduce any disulfide bridges that may have formed between the particles. After stirring for 90 min, particles were washed twice by centrifugation (10 min, 9000 rpm) and redispersed in phosphate buffer. Then, sulfo-MBS solution (20 µL, 3.75 mM) was added to the mixture. The suspension was stirred for 1 h before being purified twice by centrifugation (10 min, 9000 rpm) and redispersion in phosphate buffer. Finally, goat anti-rIgG solution (15 µL, 1.2 mg/mL) was added. After 90 min at room temperature, solid BSA was added to a final concentration of 0.5% (w/v) to block the non-specific adsorption sites, and the suspension was stirred for 45 min before the final washing step (see below).

Chemisorption to AuNR@SiO 2 -TEBA: Goat anti-rIgG solution 15 µL, 1.2 mg/mL) was added to a suspension of AuNR@SiO 2 -TEBA ([AuNR@SiO 2 ] = 15.7 pM, OD max ~ 0.6, ~ 1.5 mL in 5 mM phosphate pH 7.4). Then NaBH 3 CN solution (20 µL, 75 mM) was added. The suspension was stirred for 17 h at room temperature. Then, solid BSA was added to a final concentration of 0.5% (w/v) to block the non-specific adsorption sites. The mixture was stirred for another 45 min before the final washing step (see below).

UV-visible spectra were recorded after each step and the three protocols described above were applied to (i) FITC-labeled goat anti-rIgG to quantify the amount of Ab per nanoparticle following our previously published procedure [START_REF] Zhang | Direct quantification of surface coverage of antibody in IgG-Gold nanoparticles conjugates[END_REF] (see supplementary data for the preparation of FITC-labeled goat anti-rIgG ), and (ii) to affinity-purified goat anti-rabbit IgG (Ab) to prepare the nanoprobes.

Washing procedure: The particles were washed four times by centrifugation for 10 min at 9000 rpm. After each washing, supernatants S1 to S4 were collected and the pellets were redispersed in phosphate buffer containing BSA 0.25% w/v (1.5 mL). Supernatants S1 to S4 were centrifuged for another 15 min at 15,000 rpm in order to discard remaining particles which could interfere with the fluorescence measurements. Indirect quantification of FITClabeled goat anti-rIgG grafted to AuNR@SiO 2 was performed from fluorescence measurements of S1 to S4 (supplementary data).

Evaluation of the performances of LSPR nanobiosensors in solution

Aliquots of rabbit IgG solution (4 µL, 2.5 mg/mL) were added to the three nanoprobe suspensions AuNR@SiO 2 -Ab, AuNR@SiO 2 -MPTMS-Ab and AuNR@SiO 2 -TEBA-Ab (23.7 pM, OD max ~ 0.9, 400 µL) in plastic cuvettes, giving a final concentration between 25 and 1000 µg/mL. The mixtures were rapidly vortexed (~1 sec) after each addition. Extinction spectra were measured after one hour with a data interval of 0.3 nm.

Results and Discussion

Starting from core-shell AuNR@SiO 2 having an aspect ratio of 2.6 ± 0.2 and a silica shell thickness of 3.6 ± 0.2 nm, we elaborated three nanoprobes relying on three different surface chemistries: physisorption and, chemisorption via either thiol-terminated (MPTMS) or aldehyde-terminated (TEBA) silane condensation (Figure 1). In what follows, we first investigate the surface functionalization of AuNR@SiO 2 by silane condensation, related to the chemisorption strategy.

Surface functionalization of AuNR@SiO 2

In the chemisorption strategy, prior to conjugate the antibody to the nanoparticles, thiol or aldehyde functional groups were introduced at the surface of the silica shell through silanol condensation with alkoxysilanes MPTMS and TEBA, respectively. Nanoparticle suspensions were characterized by UV-Vis spectroscopy, DLS, ELS and TEM (Figure 2, Table 1). DLS is used in this work to evaluate the colloidal stability of the nanoparticles after grafting and not for size measurement (see experimental part for details), for which we rely on TEM images that provide more accurate data. Extinction spectra of AuNR@SiO 2 before and after silane condensation were normalized to visualize the displacement of the longitudinal plasmon band (l-LSPR) position and width. As shown in Figure 2a and Table 1, the l-LSPR band position is red-shifted by 12 nm and 4 nm after MPTMS and TEBA grafting, respectively. This shift is due to the change of the dielectric properties at the vicinity of the nanoparticles. The shift is larger for MPTMS, which might result from a larger density of MPTMS groups at the AuNR@SiO 2 surface. Moreover, asymmetric broadening of the l-LSPR band is noticed after silane condensation (Table 1, FWMH values) from 87 to 113 and 103 nm for MPTMS and TEBA, respectively. This broadening could be attributed to the coupling of plasmonic particles. No significant change in the hydrodynamic dimensions after alkoxysilane grafting was noticed from DLS measurements (Figure S 1c). One can however notice a moderate decrease in the relative intensity of the signal at ca. 13 nm associated to rotational diffusion for AuNR@SiO 2 -MPTMS sample. The decrease in intensity of the peak associated to rotational diffusion, without a significant shift of the one associated to translational diffusion, could be assigned, based on the work of Liu et al. [START_REF] Liu | Dynamic light scattering for gold nanorod size characterization and study of nanorod-protein interactions[END_REF], to the formation of small (made of 3-4 NRs) aggregates for this sample (consistent with the significant broadening of its l-LSPR band), that will not compromise the colloidal stability of the nanoparticles. Zeta potential measurements show a negative surface charge of the particles at pH 8-9 which ensures electrostatic repulsion between the particles. The zeta potential values are only slightly modified after silanes grafting (-49.1 vs -46.3 mV (MPTMS) and -44.3 mV (TEBA), Figure 2b, Table 1) reflecting the persistence of silanol groups after nanoparticle functionalization. Indeed, the evolution of the surface charge upon silane grafting depends on the surface coverage of silane and on the functional groups in the alkoxysilane molecule: for instance, Abadeer et al. [START_REF] Abadeer | Distance and Plasmon Wavelength Dependent Fluorescence of Molecules Bound to Silica-Coated Gold Nanorods[END_REF] observed the neutralization of the surface after grafting of 3azidopropyltrimethoxysilane while Kang et al. [START_REF] Kang | Interactions between Silica-Coated Gold Nanorod Substrates and Hydrophobic Analytes in Colloidal Surface-Enhanced Raman Spectroscopy[END_REF] found that the potential remains negative after chlorotrimethylsilane grafting whatever the pH. In the current case, the surface charge of particles decorated with sulfhydryl groups was slightly closer to the initial charge in comparison to the silica surface grafted with aldehyde groups. This could be explained by the fact that several silanols remain unbound to the silanes [START_REF] Aissaoui | Silane Layers on Silicon Surfaces: Mechanism of Interaction, Stability, and Influence on Protein Adsorption[END_REF][START_REF] Ben Haddada | Optimizing the immobilization of gold nanoparticles on functionalized silicon surfaces: amine-vs thiol-terminated silane[END_REF] and that the sulfhydryl group is ionizable (pK a = 8-9) whereas the aldehyde group is neutral in the whole pH range.

TEM images of AuNRs@SiO 2 before and after silane condensation (Figure 2c-e) suggest a moderate increase of the silica shell thickness from 3.6 nm to about 4.5 nm (Table 1), in agreement with the literature [START_REF] Inose | Development of composite nanoparticles composed of silica-coated nanorods and single nanometer-sized gold particles toward a novel X-ray contrast agent[END_REF]. This result is crucial since the LSPR immunosensor sensitivity depends on the silica shell thickness [START_REF] Song | Amplifying the signal of localized surface plasmon resonance sensing for the sensitive detection of Escherichia coli O157:H7[END_REF]. Furthermore, no formation of silica nanoparticles from homogeneous polycondensation of silanes was observed as shown on the TEM images of the as-synthesized samples before washing (Figure S 1a,b). This proves the selective and exclusive functionalization of the surface of AuNR@SiO 2 by silanes condensation on the silanol groups under the chosen conditions. XPS analysis was performed to evaluate the surface chemical composition of AuNR@SiO 2 , AuNR@SiO 2 -MPTMS and AuNR@SiO 2 -TEBA, and highlight the grafting. Due to the large nanoparticles size compared to the XPS analysis depth (about 10 nm), it is possible to consider the analyzed samples as a bulk material (see supplementary data for detailed calculations). The survey XPS spectra (Figure S 2) show the presence of the same elements (O, S, C, and Si) on the particles surface before and after silanes grafting.

In order to discriminate the different chemical environments of sulfur, deconvolution of the S 2p peak was performed (Figure 3). Two contributions, with their main peaks at ~164.4 eV and ~163.0 eV assigned to thiol (-SH) and gold-bound sulfur (S-Au), respectively, were identified on the high resolution S 2p spectra for AuNR@SiO 2 , AuNR@SiO 2 -MPTMS, and AuNR@SiO 2 -TEBA. The S-Au bonds are attributed to the interaction of MPTMS, used as primer, with AuNR surface. For the AuNR@SiO 2 sample, the thiol contributionsulfur not bound to gold -(Figure 3a) could be assigned to MPTMS molecules self-condensed or cocondensed with TEOS and trapped into the silica shell [START_REF] Aissaoui | Silane Layers on Silicon Surfaces: Mechanism of Interaction, Stability, and Influence on Protein Adsorption[END_REF][START_REF] Boujday | Amorphous Silica as a Versatile Supermolecular Ligand for NiII Amine Complexes: Toward Interfacial Molecular Recognition[END_REF]. The addition of MPTMS to AuNR@SiO 2 resulted in a clear increase of this contribution (Figure 3b) while it remained unchanged upon reaction with TEBA (Figure 3c). Besides these sulfur contributions, a third contribution at higher binding energy (~ 169.0 eV) assigned to oxidized sulfur forms (SO 4 ), was observed exclusively after silane condensation on the samples for both AuNR@SiO 2 -MPTMS and AuNR@SiO 2 -TEBA (Figure 3b). We first checked that this oxidized sulfur contribution did not originate from impurities in the silane samples and did not observe any oxidize sulfur on the S2p spectra recorded for MPTMS and no sulfur at all for TEBA (Figure S 3A). Another explanation could be the oxidation of free thiol that may occur in the presence of dissolved oxygen in the reaction medium [START_REF] Bagiyan | Oxidation of thiol compounds by molecular oxygen in aqueous solutions[END_REF]. To check whether this happened we applied the silane condensation conditions to bare AuNR@SiO 2 and measured the S2s and S2p peaks after heating in a EtOH/water mixture. The high resolution XPS spectra depicted in Figure S 3B clearly show the oxidation of sulfur during this process and unveil the origin of the oxidized sulfur observed above.

The XPS atomic ratios, calculated from the elemental analysis (Table S 1), further confirmed the grafting of MPTMS and TEBA owing to the significant increase in the Si/Au ratios and, for MPTMS, S/Au ratio. Note that, according to the Si/Au ratio, the amount of MPTMS appeared to be much larger than that of TEBA since it only increased from 0.97 to 1.08 in the latter case, versus 2.75 in the former case. All the same, S/Au ratio increased significantly upon MPTMS grafting (from 0.15 to 1.13, i.e. approx. 10 times higher) evidencing the presence of extra thiol groups and hence confirming the grafting of MPTMS. XPS analysis does not distinguish between the surface-exposed and the trapped thiol functions inside the silica shell, induced by the primer used to grow the silica shell. Therefore, a colorimetric assay was used to quantify the free thiol groups at the surface of AuNR@SiO 2 -T . T q w y E ' g described in section 2.3. The average surface density estimated from the experimental measurements is 2.4 ± 0.6 SH/nm 2 (n=3) (Table S 2). Unfortunately, the aldehyde group density on the surface could not be measured by such assays.

Antibodies bioconjugation to core-shell nanoparticles

AuNR@SiO 2 were bioconjugated to goat rabbit anti-IgG antibody (anti-rIgG), according to two main approaches: physisorption to bare AuNR@SiO 2 and chemisorption to the functionalized AuNR@SiO 2 -MPTMS and TEBA (Figure 1). Grafting of anti-rIgG by physisorption is based on the electrostatic interactions between the negatively charged surface of AuNR@SiO 2 and the basic amino acids of Ab (bioconjugate A). Chemisorption of anti-rIgG to AuNR@SiO 2 -MPTMS was carried out using m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS) as coupling agent. The latter reacts with the free thiol function of MPTMS via its maleimide function to form stable thioether bond and with free primary amine functions of anti-rIgG via its Nhydroxysulfosuccinimide ester group to form amide bonds (Figure S 5; bioconjugate B). Chemisorption of anti-rIgG to AuNR@SiO 2 -TEBA was performed by reductive amination between the aldehyde group of TEBA and the primary amine functions of anti-rIgG in the presence of sodium cyanoborohydride to reduce the intermediate Schiff base (Figure S 5; bioconjugate C). A blocking step to prevent non-specific adsorption was performed using bovine serum albumin (BSA) for all the bioconjugates. The nanoparticles were analyzed after each synthesis step by UV-Visible spectroscopy as shown in Figure 4. All the bioconjugations were performed in triplicate and the position of the l-LSPR band (determined from the zero-crossing of the first order derivative using the 19points method [START_REF] Chen | Inflection Point of the Localized Surface Plasmon Resonance Peak: A General Method to Improve the Sensitivity[END_REF]) and its width are given in Table S 3 (mean and standard deviations calculated from three replicates). The l-LSPR band of AuNR@SiO 2 , initially at 716.2 nm, red-shifted by 8.8 nm after addition of anti-rIgG, which proves its adsorption onto nanoparticles surface (Figure 4 a). Further red shift of 5.8 nm is noted after blocking step with BSA. The l-LSPR band of AuNR@SiO 2 -MPTMS is progressively red-shifted by +3.0, +5.6 and +5 nm after reaction with sulfo-MBS, anti-rIgG grafting and blocking step with BSA, respectively. Similar red-shifts of +2.5, +7.1 and +5.2 nm were observed for AuNR@SiO 2 -TEBA following anti-rIgG grafting and BSA adsorption respectively. These results show that physisorption of antibody induces the largest shift of the l-LSPR band followed by chemisorption via TEBA and MPTMS, without significant increase of the FWHW. Figure S 6 (top) show an increase in the hydrodynamic diameter from about 110 nm to about 150 nm upon chemisorption of anti-rIgG to AuNR@SiO 2 -MPTMS/TEBA while the size of the physisorption-based bioconjugate was unchanged. Figure S 6 (bottom) illustrates that bioconjugation increases the zeta potential from about -30/-27 mV to around -12/-16 mV in phosphate buffer. This result is expected as the nanoparticles surface charge should be close to that of the adsorbed protein [START_REF] Zhang | Direct quantification of surface coverage of antibody in IgG-Gold nanoparticles conjugates[END_REF]. These values are consistent with those previously measured for bioconjugated spherical gold [START_REF] Ben Haddada | Gold nanoparticle-based localized surface plasmon immunosensor for staphylococcal enterotoxin A (SEA) detection[END_REF] and Au@Ag core-shell [START_REF] Loiseau | Core-Shell Gold/Silver Nanoparticles for Localized Surface Plasmon Resonance-Based Naked-Eye Toxin Biosensing[END_REF] nanoparticles.

DLS measurements in

The amount of anti-rIgG grafted to the nanoparticles was indirectly estimated by measuring the concentration of the unreacted FITC-labeled anti-rIgG by spectrofluorimetry, as previously described [START_REF] Zhang | Direct quantification of surface coverage of antibody in IgG-Gold nanoparticles conjugates[END_REF] and detailed in the supplementary data section. The surface coverage expressed as anti-rIgG/nanoparticle ratio was estimated to 135 for AuNR@SiO 2anti-rIgG (bioconjugate A), 238 for AuNR@SiO 2 -MPTMS-anti-rIgG (bioconjugate B) and 262 for AuNR@SiO 2 -TEBA-anti-rIgG (bioconjugate C) (Figure 4 d) on the basis of three independent measurements. The theoretical antibody surface coverages estimated from the size of IgG (12x17x7 nm 3 ) and three possible orientations of the antibody molecules with respect to the nanoparticle surface [START_REF] Abadeer | Distance and Plasmon Wavelength Dependent Fluorescence of Molecules Bound to Silica-Coated Gold Nanorods[END_REF] are also plotted in Figure 4d for comparison. The experimental surface coverage of bioconjugate A appeared to match the theoretical one. In contrast, the coverage values for bioconjugates B and C were about twice higher than the theoretical ones. It could be concluded from this result that the lower coverage of reactive functions in the case of AuNR@SiO 2 -TEBA does not affect the amount of adsorbed anti-rIgG identical to that obtained for AuNR@SiO 2 -MPTMS. The reason is probably because in the case of chemisorption, both strategies involve reaction with antibody amine groups, however, the number on anchoring points may affect the orientations of antibodies. The lower coverage obtained in the case of physisorption reflects the formation of a less dense antibody layer at the surface of the silica shell, that may also reflect a different orientation of the antibody molecules.

Evaluation of the LSPR nanobiosensors in solution

AuNR@SiO 2 bioconjugated to affinity-purified goat anti-rabbit IgG antibody (Ab), hereafter named nanoprobes A, B and C, were synthesized accordingly used for the LSPR immunosensing of rabbit IgG in solution. Three nanoprobes were synthesized by the three surface chemistries described above: Ab physisorption for AuNR@SiO 2 (nanoprobe A) and Ab chemisorption for AuNR@SiO 2 -MPTMS/TEBA (nanoprobes B and C, respectively). Transduction of the biorecognition is based on the l-LSPR band shift owing to RI change at proximity to the nanoparticles surface.

Response to rabbit IgG

A preliminary experiment was performed to assess the ability of the nanoprobes to sense the target antigen, namely rabbit IgG, via the shift of their l-LSPR band. The three nanoprobe suspensions were supplemented with 1000 ng/mL rIgG, the extinction spectrum of the mixtures was recorded at 10 min time interval and the shifts of the l-LSPR peak maximum (determined from the first derivative spectra, see below) were plotted as a function of time (Figure S 7). A progressive red shift of the l-LSPR band was observed until 40 min whose magnitude depended on the nanoprobe (A > B > C). This evidenced that the three nanoprobes were indeed able to optically detect the target in a short time scale. Interestingly, probes B and C provided the same response (∆ max ) for 1000, 750 and 500 ng/mL rabbit IgG after 1 h incubation (Figure S 8, Table S 4) as a result of nanoprobe saturation from 500 ng /mL.

Next, the nanoprobe suspensions were supplemented with increasing concentrations of rabbit IgG ranging from 25 to 500 ng/mL and extinction spectra were recorded one hour after each new addition. The extinction spectra of nanoprobe A and their respective first and second derivatives are presented in Figure 5 (for nanoprobes B and C, see Figures S 9 and S 10, respectively). The maximum of the l-LSPR peak (Point 2) and the inflection point at the long wavelength side (Point 3) are defined by dotted lines. Point 2 is obtained from the 0 value of the first order derivative spectrum while Point 3 is obtained from the 0 value of the second order derivative spectrum. A progressive shift of the plasmon band is observed subsequently to the addition of increasing amounts of rabbit IgG for all the nanoprobes. The corresponding values of the tracking points 2 and 3 are summarized in Table 2. Displacements of up to 4.9, 4.2 and 2.4 nm were determined from point 2 and 7.6, 9.3 and 3.0 nm were determined from point 3 for AuNR@SiO 2 -Ab (nanoprobe A), AuNR@SiO 2 -MPTMS-Ab (nanoprobe B) and AuNR@SiO 2 -TEBA-Ab (nanoprobe C), respectively. Note that these displacements are smaller than those recorded after antibodies adsorption during bioconjugate engineering, possibly because the distance to the plasmonic surfaces are bigger. The three nanoprobes gave shifts that did not significantly differ from those of the blanks. This proves the specificity of the nanoprobes for rabbit IgG detection.

Stability and analytical performances

DLS measurements (Figure S 11) show that nanoprobes maintain a good colloidal stability after rabbit IgG capture. This result dramatically differs from previous works dealing with solution-phase AuNR-based LSPR biosensors for which addition of target resulted in nanoparticles aggregation that translated into large redshifts and drastic reduction of plasmon band intensity [START_REF] Guo | Fabrication of Antihuman Cardiac Troponin I Immunogold Nanorods for Sensing Acute Myocardial Damage[END_REF][START_REF] Wang | Biorecognition-Driven Self-Assembly of Gold Nanorods: A Rapid and Sensitive Approach toward Antibody Sensing[END_REF][START_REF] Wang | Gold Nanorod Probes for the Detection of Multiple Pathogens[END_REF]. The l-LSPR band shifts ( LSPR ) measured at points 2 and 3 were plotted as a function of rabbit IgG concentration (Figure 6). Non-linear regression of the data was performed according to the 4-parameter logistic equation as detailed in supplementary data section. The analytical performances of the three nanoprobes (IC 50 , dynamic range and limit of detection (LoD)) as well as the parameters attesting the accuracy of the model (R 2 ,   ) are summarized in Table 3. The LoD is defined as the concentration corresponding to 10 % of the maximum response (IC 10 ) [START_REF] Soares | Adsorption according to the Langmuir-Freundlich model is the detection mechanism of the antigen p53 for early diagnosis of cancer[END_REF]. The dose-response curves show similar trends for nanoprobes A and B and clearly lower performances for nanoprobe C. In all cases, monitoring the wavelength of the second derivative inflection point gave a larger response that following the maximum of l-LSPR position. The LoD were estimated to 27 ng/mL, 12 ng/mL and 31 ng/mL (80 -207 pM) for nanoprobes A, B and C respectively (point 3). The LoD of nanoprobes A and B were about 3 to 4-fold lower than those measured with closely related systems for the detection of human IgG (0.4 nM) [START_REF] Wang | Biorecognition-Driven Self-Assembly of Gold Nanorods: A Rapid and Sensitive Approach toward Antibody Sensing[END_REF], which proves their excellent analytical performances. Altogether, the three LSPR nanobiosensors had excellent analytical performances with narrow dynamic ranges and the lowest LoD equal to 12 ng/mL. The best analytical performances are provided by nanoprobe B, built on AuNR@SiO 2 -MPTMS surface, in terms of LoD, IC 50 , and dynamic range. This chemisorption strategy that induced a high coverage of reactive functions, obviously led to efficient nanoprobes with accessible and well-oriented antibodies. Nanoprobe A, that was built by physisorption of anti-rIgG also exhibits remarkable performances, close to those of nanoprobe B. T " p p y p gy" p y y g y g g that leads to strong electrostatic interactions. Beside these electrostatic interactions, silanol groups, present with high density (~5 Si-OH/nm²) are easily involved in H bonds with amine groups [START_REF] Aissaoui | Silane Layers on Silicon Surfaces: Mechanism of Interaction, Stability, and Influence on Protein Adsorption[END_REF]. They certainly provide cooperative interactions upon anti-rIgG physisorption further stabilizing the bioconjugate. In addition, for an LSPR biosensor to be efficient, binding of analyte should occur within the sensing volume that is determined by the decay length. In the case of nanoprobe A, the bioreceptor is the closest to the gold surface which gives rise to larger LSPR peak shifts and further improves its performances. Nanoprobe C gave much lower responses and a comparatively narrower dynamic range than nanoprobes A and B, possibly because of an unfavorable orientation of the capture antibody that hinders proper antigen binding or a limited accessibility of the bioreceptors. The low coverage of silanes during the preparation of AuNR@SiO 2 -TEBA may be one of the reasons to these lower performances even if electrostatic interactions should have compensated the reactivity. Nevertheless, the difference observed between nanoprobes B and C, despite identical amounts of bioreceptors on their surface, clearly highlights the importance of the surface chemistry on bioreceptor orientation and nanoprobes efficiency for biosensing.

Conclusion and perspectives

We have reported here an in-depth investigation of the biofunctionalization of gold nanorods coated with an ultrathin layer of silica (AuNR@SiO 2 ) to elaborate LSPR nanobiosensors for solution-phase biosensing of proteins. Three surface chemistries were explored to attach the capture antibody at the surface of AuNR@SiO 2 , the first via physisorption to the negatively charged silica-coated nanorods and the second and third by first condensing reactive silane terminated by thiol (MPTMPS) or aldehyde (TEBA) to the silica layer, then cross-reacting with the antibodies amino-groups to achieve a stable chemisorption. Each step of surface functionalization was characterized with DLS, electron microscopy, zetametry, XPS and UV-Visible spectroscopy. Silane grafting was achieved with higher rate in the case of MPTMS than for TEBA, but the colloidal stability was preserved for both routes. The coverage rate of immobilized antibodies was estimated by fluorimetric analysis of the supernatants and was twice lower for the physisorption bioconjugate while identical for the two chemisorbed bioconjugates despite the differences in silane coverage. The resulting nanoprobes were efficient for the detection of the target analyte; the LSPR peak of the three nanoprobes shifted to the red and the shifts were related to the concentration between 25 and 500 ng/mL. These shifts could be readily measured with a benchtop UV-Visible spectrometer without the need of a separation step. The three probes were also highly specific since no shift was observed for an unrelated analyte. The nanobiosensors maintain an excellent colloidal stability after analyte recognition and their analytical performances were extremely high with an LoD down to 12 ng/mL in the case of the chemisorbed antibodies on MPTMS-modified AuNR@SiO 2 . As a perspective, the LSPR response could be further optimized by choosing AuNR whose dimensions determine a decay length that matches the thickness of the bioreceptoranalyte couple. Another perspective of signal improvement could be the reduction of the bioreceptor size (that is the thickness of the bioreceptor layer) to maximize the occupation of the sensing volume.

Appendix A. Supplementary data

Supplementary Figures:

Supplementary Tables:

Table S 1. XPS atomic ratios of chemical elements for bare and silanized AuNR@SiO 2 Table S 2. Assay of surface thiols by DTNB on three batches of AuNR@SiO2 silanized by MPTMS Table S 3. Summary of l-LSPR band positions and FWMH for conjugated AuNR@SiO 2 -Ab (bioconjugate 1), AuNR@SiO 2 -MPTMS-Ab (bioconjugate 2), AuNR@SiO 2 -TEBA-Ab (bioconjugate 3). Data are presented as mean ± SD (n =3) Table S 4. Shifts of l-LSPR peak maximum of nanoprobes A, B and C upon addition of rIgG solution (500 -1000 ng/mL)

Figure 1 .

 1 Figure 1. Sequences of reactions leading to AuNR@SiO 2 -Ab bioconjugates A-C or nanoprobes A, B and C. Bioconjugation of goat anti-rabbit IgG antibody (Ab) to AuNR@SiO 2 by (a) physisorption and (b, c) chemisorption via MPTMS and TEBA condensation, respectively.

Figure 2 .

 2 Figure 2. Characterization of AuNR@SiO 2 before (gray) and after silica shell functionalization by MPTMS (green) or TEBA (blue). (a) normalized extinction spectra, (b) zeta potential distribution at pH 8-9 and (c-e) representative TEM images.

Figure 3 .

 3 Figure 3. S 2p XPS spectra of AuNR@SiO 2 before (a) and after silane condensation with (b) MPTMS and (c) TEBA

Figure 4 .

 4 Figure 4. Normalized UV-visible spectra and their first derivatives (insets) recorded after the successive steps schematized in figure 1. (a) bioconjugate A (gray traces), (b) bioconjugate B (green traces), (c) bioconjugate C (blue traces); (d). Estimated (full bars) and theoretical (hatched bars) antibody surface coverage for bioconjugates 1, 2 and 3. Data are presented as mean ± SD (n =3).

Figure 5 .

 5 Figure 5. Normalized UV-visible spectra (top), first derivative (middle) and second derivative (bottom) ag of nanoprobe A (physisorption) after exposure to different concentrations of rabbit IgG (0, 25, 50, 100, 150, 250 and 500 ng/mL). Inset: focus on the zero values of the first and second derivatives showing the l-LSPR band shifts at point 2 ( max ) and point 3 (inflection point), respectively.

Figure 6 .

 6 Figure 6. LSPR nanobiosensors dose-response curves obtained by plotting point 2 (squares) or point 3 (dots) vs. rabbit IgG concentration for nanoprobe A (left), nanoprobe B (middle) and nanoprobe C (right). Data are presented as mean ± SD (n =2). Curves result from non-linear fitting of data according to 4-parameter logistic equation.

Figure S 1 .

 1 Figure S 1. TEM images of AuNR@SiO 2 silanized with (a) MPTMS and (b) TEBA before washing step (c) Hydrodynamic diameter of silanized AuNR@SiO 2 samples after washing Figure S 2. Survey XPS spectra of AuNR@SiO 2 before (black) and after reaction of MPTMS (red) and TEBA (blue).

Figure S 3 .

 3 FigureS 3. XPS investigation of the origin of oxidized sulfur in the functionalized AuNR@SiO 2 . Panel A shows S2p spectra for the reagents silanes, MPTMS and TEBA, the latter contains no sulfur and Panel B shows the S2s and S2p spectra for AuNR@SiO 2 before and after thermal treatment in EtOH/H2O mixture without adding any silane, this treatment clearly affects the oxidation state of sulfur and new species at higher binding energy appear on S2p peak Figure S 4. UV-Vis spectra of DTNB (green) and supernatants of silanized (red) and non-silanized (black) AuNR@SiO2 samples after reaction with DTNB.

Figure S 5 .

 5 Figure S 5. General scheme for grafting of anti-rabbit IgG to AuNR@SiO2 via (a) surface thiol functions and (b) surface aldehyde functions Figure S 6. DLS (top) and zeta potential measurements (bottom) of AuNR@SiO 2 (-MPTMS/TEBA) before and after bioconjugation and non-specific blocking step with BSA at pH 7.4.

Figure S 7 .Figure S 8 .

 78 Figure S 7. Shift of the l-LSPR peak position over time for AuNR@SiO2-Ab (nanoprobe A), AuNR@SiO2-MPTMS-Ab (nanoprobe B), and AuNR@SiO2-TEBA-Ab (nanoprobe C) in the presence of rabbit IgG antigen (1 µg/mL) Figure S 8. Normalized extinction spectra (top) and their first derivatives (bottom) of nanoprobes after addition of rabbit IgG in the range 500-1000 ng/mL. AuNR@SiO 2 -Ab (nanoprobe 1, gray traces). (B) AuNR@SiO 2 -MPTMS-Ab (nanoprobe B, green traces) and (C) AuNR@SiO 2 -TEBA-Ab (nanoprobe C, blue traces) Figure S 9. Normalized UV-visible spectra (top), first derivative (middle) and second derivative (bottom) of AuNR@SiO 2 -MPTMS-Ab (nanoprobe B) after exposure to different rabbit IgG concentrations (0, 25, 50, 100, 150, 250 and 500 ng/mL) Figure S 10. Normalized UV-visible spectra (top), first derivative (middle) and second derivative (bottom) of AuNR@SiO 2 -TEBA-Ab (nanoprobe C) after exposure to different rabbit IgG concentrations (0, 25, 50, 100, 150, 250 and 500 ng/mL) Figure S 11. DLS measurements before and after addition of rabbit IgG for (a) AuNR@SiO 2 -Ab (nanoprobe A), (b) AuNR@SiO 2 -MPTMS-Ab (nanoprobe B) and (c) AuNR@SiO 2 -TEBA-Ab (nanoprobe C) Figure S 12. Normalized UV-visible spectra (left), first derivative (middle) and second derivative (right) of the nanoprobes after exposure to solutions of non-specific mouse IgG (0, 1 or 2 µg/mL); (a) AuNR@SiO2-Ab (nanoprobe A) (b) AuNR@SiO2-MPTMS-Ab (nanoprobe B) and (c) AuNR@SiO2-TEBA-Ab (nanoprobe C)

Table 1 .

 1 Summary of AuNR@SiO 2 properties before and after silane condensation.

	Sample	 LSPR (nm) / (nm)	FWMH (nm)	Zeta potential (mV)	@SiO 2 (nm) (TEM)	@SiO 2 (nm) (XPS)
	AuNR@SiO 2	716/ -	87	-49 ± 1	3.6 ± 0.6	2.12
	-MPTMS	728/12	113	-46 ± 1	4.7 ± 1.2	4.21
	-TEBA	720/4	103	-44 ± 2	4.5 ± 0.9	2.53

Table 2 .

 2 Summary of l-LSPR band shifts ( LSPR ) for nanoprobes A, B and C as a function of rabbit IgG concentration according to points 2 and 3; the shift of the l-LSPR band after addition of mouse IgG (mIgG) is also reported to ascertain the specificity of the nanoprobes. Data are given as mean ± SD (n=2) specific recognition; b nonspecific protein; c standard deviation of the blank samples Larger shifts are observed from point 3 because it takes into account both the position of the LSPR peak maximum and its broadening[START_REF] Chen | Inflection Point of the Localized Surface Plasmon Resonance Peak: A General Method to Improve the Sensitivity[END_REF]. Band broadening could be due to nanoparticles cross-linking via rabbit IgG, nevertheless no color change is noticed for the colloidal solutions. The specificity of the nanoprobes was also checked by adding mouse IgG instead of rabbit IgG at two concentrations (1 and 2 µg/mL, values in Table2, spectra in FigureS 12).

					[rIgG] a (ng/mL)			[mIgG] b (ng/mL)
		0	25	50	100	150	250	500	1000	2000
	Nanoprobe	Pt 2 0.03 c 0.3 ± 0.2 0.9 ± 0.7 2.6 ± 0.4 3.8 ± 0.9 4.9 ± 0.2 4.9 ± 0.2 0.09	0.17
	A	Pt 3 0.45 0.7 ± 0.2 2.3 ± 0.7 4.6 ± 0.4 6.5 ± 0.2 7.6 ± 0.2 0.66	0.33
	Nanoprobe	Pt 2 0.08 0.30 ±	0.50 ±	1.7 ± 0.1 2.7 ± 0.1 3.7 ± 0.2 4.2 ± 0.2 -0.25	-0.06
	B		0.03	0.05
		Pt 3 0.46 2.4 ± 0.2 2.8 ± 0.3 4.8 ± 0.2 6.5 ± 0.3 8.5 ± 0.4 9.3 ± 0.5 1.72	0.84
	Nanoprobe	Pt 2 0.02 0.10 ±	0.6 ± 0.1 1.8 ± 0.2 2.4 ± 0.2 2.5 ± 0.2 2.4 ± 0.2 0.025	0.03
	C		0.01
		Pt 3 0.24 0.05	1.0 ± 0.1 2.2 ± 0.2 2.7 ± 0.3 3.2 ± 0.3 3.1 ± 0.3 0.34	0.35

a

Table 3 .

 3 Analytical performances and fitting parameters for LSPR nanobiosensing of rabbit IgG with nanoprobes A, B and C

		Nanoprobe A	Nanoprobe B	Nanoprobe C
		point 2	point 3	point 2	point 3	point 2	point 3
	∆ max	5.1 ± 0.2	7.9 ± 0.3	4.4 ± 0.1	12 ± 2	2.51 ± 0.07	3.2 ± 0.1
	IC 50 (ng/mL)	98 ± 6	80 ± 6	125 ± 5	120 ± 50	73 ± 4	70 ± 6
	 2	0.082	0.200	0.019	1.05	0.029	0.065
	R 2	0.997	0.997	0.999	0.985	0.996	0.994
	LoD (ng/mL) a	38	27	43	12	36	31
	DR (ng/mL) b	54 -156	40 -140	64 -207	26 -206	46 -105	41 -117
	a calculated from 10% of ∆ max ; b dynamic range (DR) corresponding to concentrations giving from 20
	to 80% of ∆ max

Acknowledgement

This work was financially supported by the French National Research Agency (Project No. ANR-16-CE07-0031-01) y ' 'A , France

Authors contribution

Conceptualization & Methodology: SB & MS; Investigation: VP, JB, AM, CM, SMC; Writing -Original Draft: FS; Writing -Review & Editing: SB & MS; Supervision: SB, MS, JB; Funding acquisition: SB.