Nanomaterials and especially plasmonic nanoparticles are more and more studied in the biomedical field. This increasing interest lies in their remarkable chemical and optical properties resulting from their nanosize. In particular, gold nanoparticles (AuNPs) exhibit a localized surface plasmon resonance (LSPR) band in the visible region, which strongly depends on the dielectric properties of their local environment. In addition, several chemistries are known that allow their functionalization with a large variety of biomolecules (peptides, proteins, DNA, polymers, ...). These features gave rise to numerous developments as nanocarriers for in vivo drug delivery, hotspots for phototherapy, contrast agents for imaging systems, or colorimetric reporters for IVD. For all these applications, AuNPs must be engineered to (i) possess sufficient chemical and colloidal stabilities and (ii) express specificity for target molecules or tissues. Thiol chemistry is usually used to functionalize the particles as it is a simple and convenient strategy. However, thiol chemistry possesses two severe drawbacks limiting their use for in vivo applications: (i) a limited chemical robustness of the organic layer due to the lability of the Au−S bond and (ii) the difficulty to control the grafting density of different thiolated molecules on the AuNP surface.1 A new a nanoparticle functionalization strategy taking advantage of calix[4]arene-tetradiazonium salts was recently developed (Figure 1).2 It has been shown that thin layers of these molecular platforms can be strongly and irreversibly grafted to the NP surface via the reduction of their diazonium groups that leads to the formation of multiple covalent bonds. Furthermore, as calix[4]arene-tetradiazonium salts differently substituted on the phenol positions possess a similar reactivity for gold surfaces, it was shown that the composition of mixed layers of calix[4]arenes could be controlled on AuNPs3, further allowing to control the grafting density of different biomolecules on their surface.4 This strategy could further be extended to synthesize silver and alloyed gold-silver nanoparticles,5 displaying high colloidal and chemical stabilities, despite the thinness of the organic layer, and a controlled bioconjugation capacity. These silver nanoparticles could be functionalized with peptide aptamers and used to detect targets of biological interest (the Mdm2 oncoprotein, anti-SARS-CoV-2 antibodies) using several colorimetric strategies, i.e. Lateral Flow and turbidimetry assays.6 We do believe that this strategy could pave the way to the development of many other biosensing systems based on AgNPs coated with a calixarene layer as robust and efficient colorimetric reporters. 1. Retout, M., Brunetti, E., Valkenier, H., Bruylants, G. (2019) JCIS 557, 807−815. 2. Troian-Gautier, L., Valkenier, et al. (2016) Chem. Commun. 52, 10493−10496.. 3. Valkenier, H., Malytskyi, et al. (2017) Langmuir 33, 8253. 4. M. Retout, P. Blond, I. Jabin, G. Bruylants, Bioconjugate Chem. 2021, 32, 290–300. 5. M. Retout, I. Jabin, G. Bruylants, ACS Omega 2021, 6, 19675–19684. 6. M. Retout, B. Gosselin, A. Mattiuzzi, I. Ternad, I. Jabin, G. Bruylants ChemPlusChem, doi.org/10.1002/cplu.202100450

We investigated a sensitive and specific ON-OFF SERS scheme for miRNA detection using a DNA origami nanoforks and gold nanoparticles (DONA) structure integrated with hairpin DNA probes on optical fibers. The biosensor is designed employing a stem-loop/hairpin DNA probe with Raman label at one end, attached at the DONA hotspot, as a target identification switch. The switch is initially kept OFF by a placeholder DNA strand which hybridizes to the hairpin probe through a specific region. In the presence of target miRNA, the placeholder strand is displaced initiated by a toehold binding and branch migration mechanism . This process finally releases the placeholder strand and the hairpin probe is closed bringing the Raman label in the SERS hotspot, enabling the switch ON. Thus, essentially the SERS signal of the Raman label turns OFF to ON in the presence of target miRNA enabling the direct detection of these biomarkers. To avoid the false signals, the gold nanoparticles are coated with an internal standard (IS) molecule to record a ratiometric SERS signal.

Coating of gold nanorods with a silica shell to generate core-shell AuNR@SiO2, is one of the most effective routes to promote their applications in the biomedical field [1]. Among other assets, the silica shell improves the nanoparticles stability, frees the surface from toxic cetyltrimethylammonium bromide (CTAB), and preserves the rod shape under photothermal conditions. When the intended application is nanoplasmonic biosensing, the silica shell needs to be very thin while a thicker and porous shell is best suited for drug delivery. Our first objective was to devise robust procedures to generate silica shells on gold nanorods having both controlled pore organization and shell thickness to generate nanoobjects adapted to the desired biological application. Our second objective was to design a localized surface plasmon resonance (LSPR) nanobiosensor from the AuNR@SiO2 for the label-free detection of a model target. By modulating the pH of the reaction medium, control over the silica growth rate and morphological parameters of the shell was achieved. Different silica shell thicknesses, from 13 to 21 nm, were produced by varying the Au/TEOS ratio. Depending on the pH set for TEOS condensation, the orientation of the silica porosity was tailored to be either perpendicular, or, more unexpectedly, parallel to AuNR surface. Finally, the use of a surface primer allowed the formation of ultrathin non porous silica shells with a thickness precisely tunable between 2 and 6 nm by varying the Au/TEOS ratio. These synthesis methods maintained the colloidal stability and the optical properties of the plasmonic core were preserved. Complete removal of CTAB from AuNR@SiO2 was demonstrated by XPS, Raman and zeta potential measurements. Moreover, the refractive index sensitivity factor of AuNR@SiO2 having a thin layer of silica was improved by 30 % compared to CTAB-capped AuNR [2]. Nanoimmunoprobes were eventually built up from AuNR@SiO2 with an ultrathin silica shell by physisorption of the antibody to the silica surface. Alternatively, antibody conjugation was achieved via silanization with aldehyde- or thiol-terminated alkoxysilanes. Label-free, solution-phase LSPR biosensing of the model target rabbit IgG was demonstrated with excellent analytical performances in terms of dynamic range and limit of detection.

Gold nanoparticles (AuNPs) are now heavily used in various biomedical applications such as gene or drug delivery as well as in biosensors. To obtain good efficacy for the delivery systems, one of the critical factors to consider is the AuNPs diffusion properties. Furthermore, since there is a growing interest in targeting systems that allow the reduction of side effects, the influence of the functionalization and coating of AuNPs on the diffusion behavior may become a limitation. Few studies investigated the diffusion of bare gold NPs in viscous environments and found a threshold under which there is no more size dependency. [1] Here, we introduce a technique to accurately measure the diffusion coefficient of AuNPs and functionalized AuNPs into viscous liquids. The method is based on the analysis of the NPs Brownian movements and the diffusion coefficients of each AuNPs are calculated by the covariance estimator (CVE) method. To represent typical biological viscous fluids, the samples were suspended into different hyaluronic acid (HA) solutions as it is one of the main components of these liquids. The AuNPs displacements were recorded thanks to the side-illumination device [2]. Data were obtained for 80-100 nm AuNPs in viscous liquids from 9.54x10-4 to 1.90 Pa.s for bare and functionalized AuNPs (PEG, PEG-Ab, HA). Diffusion coefficient values vary from 0.01 to 25µm²/s depending on the solution viscosity and do not follow the Stokes-Einstein-Debye classical relationship but the fractional one given by D=D_0\left(\frac{\eta_0}{\eta}\right)^p with η the environment viscosity, η0 the water viscosity (9.54x10-4 Pa.s) , D0=21.1µm²/s and p=0.53. Results indicate that for all functionalization investigated, the diffusion coefficient stays relatively the same as for bare AuNPs. This is probably due to the fact that the measured zeta potentials coatings for all functionalization chemistries stay more or less negative. These results suggest that for various applications using AuNPs injections in the vitreous, lymphatic fluids, or mucus which may require different targeting approaches, the choice of functionalization chemistry will not affect the AuNPs diffusion behavior. [1] D. Coglitore, S. P. Edwardson, P. Macko, E. A. Patterson, and M. Whelan, “Transition from fractional to classical Stokes-Einstein behaviour in simple fluids,” R. Soc. Open Sci., vol. 4, no. 12 (2017) [2] M. Qi, C. Darviot, S. Patskovsky, and M. Meunier, “Cost-effective side-illumination darkfield nanoplasmonic marker microscopy,” Analyst, vol. 144, no. 4 (2019)

The production of Gold nanoparticles-supplemented implants for radiotherapy and phototherapy, necessitates the development of advanced procedures enabling the optimal distribution of GNPs in biocompatible 3D printable polymer matrices. Polyetheretherketone (PEEK), a polymer featuring high mechanical properties, radiation and corrosion resistance (including to sterilisation cycles), and biocompatibility upon implantation in vivo , could be the key to the development of advanced biomedical implants enabling the treatment of cancer by a combination of radiotherapy and photothermal therapy. Therefore, this study aimed at a) producing PEEK filaments containing a homogeneous distribution of GNP over their entire volume and b) developing a high-sensitivity and resolution physico-chemical characterisation procedure for these materials. GNP-containing filaments were extruded by using medical-grade PEEK. Two types of gold nanoparticles were used: gold nanospheres (GNS) produced by the Turkevitch method (hydrodynamic diam. by DLS: 11 nm number weighted), and gold nanorods (GNR; 100 x 15 nm; aspect ratio 7; max. absorbance peaks: 1035 – 1075 nm) produced by a proprietary method by Sona Nanotech inc (Halifax, NS, Canada). The extruded filaments presented both a geometry, Au concentration, and mechanical properties adequate for use as a feed material for FFF (1.75 ± 0.1 mm diam.; 2m ± 20 cm in length; 0.30 % and 0.11 % WAu/WPEEK for GNS and GNR, respectively). Particles were uniformly distributed in the PEEK matrix, and their physico-chemical characterizationby XPS/FT-IR did not reveal major changes in the structure of the polymer (slight difference in oxygen contents observed after PEEK extrusion). PEEK filaments containing plasmonic gold nanoparticles were successfully produced for FFF 3D printing, which open possibilities for the production of functional biomedical implants (radiotherapy and photothermal therapy).