Asymmetric attachment and functionalization of plasmonic nanoparticles on ceramic interfaces
The demands for materials that integrate more than one functional imaging or therapeutic unit are of increasing interest for biomedical applications. Here, we present the step-by-step preparation of asymmetric and optically active particles, namely, Gd2O3@Ag, Gd2O3@Au, SiO2–N3@Au, and SiO2–SH@Au . Successful attachment of plasmonic nanoparticles to the surface of metal-oxide spheres without necessity of a potentially toxic inter-adhesive layer was proven by optical methods as well as X-ray photoelectron spectroscopy. The combination of optical and magnetic properties as present in Gd2O3@Ag and Gd2O3@Au Janus-type particles leads to dual-imaging probes for optical and magnetic resonance imaging. In addition, functional groups, such as azide groups, were linked to the surface of silica particles previous to Au nanoparticle attachment. Subsequent site-selective click reactions with 5-FAM were successfully performed as demonstrated by UV–Vis measurements. All described systems exhibited excellent long-term stability and can, therefore, be considered as promising candidates for theranostic applications.
KeywordsTheranostics Janus particles Optical materials Sputter deposition Magnetic
The rapid evolution of nanoprobe applications in medicine demands advanced control over their intrinsic properties, which usually rely on the composition and shape of nanoprobes, as well as their proper functional surface modification . Beside conventional preparation methods, such as solvothermal reactions, a variety of specialized protocols for nanocluster formation, e.g., using nanoreactors  are reported in the literature. In the field of biomedicine, nanoparticles (NPs) have become more and more important. Besides drug delivery applications, NPs are widely used for in vivo imaging techniques. To date, a variety of nanocolloidal systems are reported for optical bioimaging applications including rare-earth-doped nanoparticles , carbon nanodots , and metallic nanoshells . While optical methods are range limited by light absorption of the examined body, and still, in need of invasive techniques, magnetic resonance imaging (MRI) is suitable for non-invasive imaging of malignant tissues. Being well-established as a strong T1 contrast agent [6, 7], Gd2O3 NPs bare a large magnetic moment of 7.94 μB per Gd3+ ion, leading to a high longitudinal relaxivity of nearby water protons.
In general, molecular surface modifications of these particles can be performed by grafting methods , whereas biomedically useful surface modifications are mostly achieved via cycloaddition reactions, e.g., the famous click reaction [9, 10, 11], biotin–streptavidin interactions , and carbodiimide coupling reactions . By these techniques, immobilization of antibodies, drugs, and vitamin units is achieved and widely reported in the literature [14, 15, 16]. Among a broad variety of surface grafting techniques, click reactions have emerged as one of the most commonly used functionalization strategies based on their high selectivity, the tolerance of a wide range of solvents and pH and their high yield even at room temperature . The most prominent type of click reaction is the copper(I) catalyzed alkyne–azide click reaction which leads to the formation of a stable triazole ring. While the non-catalyzed Huisgen reaction produces an unspecific mixture of 1,4 and 1,5-disubstitution products, only 1,4 disubstituted 1,2,3-triazoles are formed when copper is employed as catalyst. Besides alkyne–azide coupling reactants, thiol-ene and thiol-yne click reactions offer additional copper-free pathways for surface chemistry. As an example, Zhang et al. reported a successful immobilization of boronic acid on magnetic NPs for side-selective capture of glycoproteins . In fact, it has been shown that click reactions offer larger efficiencies than their prominent carbodiimide counterparts . However, all these techniques lack a chemical side selectivity, which results in an unspecific localization of ligands on the particle surface. The attachment of two types of ligands to one particle thus still remains a major challenge. In this regard, Janus-type particles, which possess an asymmetric geometry, have found increasing attention during the last years. The combination of two material types in one particle offers beneficial physicochemical properties that can help to overcome limitations of one material. For example, an asymmetric combination of plasmonic and magnetic characteristics in one particle provides a novel platform for multimodal imaging and therapeutic applications. Simply spoken, only one injection would be needed for detection of cancer by MRI and a direct treatment of infected cells by photothermal therapy . Janus-type particles have been applied as sensors [19, 20], catalysts , emulsifiers [22, 23], and building blocks for the formation of self-assembled superstructures , reports on an asymmetric combination of plasmonic and magnetic properties for biomedical applications are rather rare . In addition, a variety of preparation methods are presented for polymeric Janus particles, as reviewed extensively [25, 26], while a controllable preparation of biocompatible ceramic NPs, baring asymmetric functionalities is still missing. Even though a simple sputtering approach of gold NPs on immobilized silica particles was already presented in 1988 by Casagrande et al. ., shell stability could only be achieved by adhesive layers, such as Ti/W alloy or Ni, at the metal-oxide interface [28, 29]. However, due to the cytotoxic properties of these metals, these interlayers lower the biomedical applicability of the particles dramatically and limit their possible application for in vivo studies. In this approach, we present a simple method for the preparation of metal-oxide particles which can be surface decorated with plasmonic silver or gold nanoparticles. Unlike the procedures reported in the literature, no metallic inter-adhesive layers are needed for the formation of long-term stable dispersions of Janus-type functionalized ceramic particles. Instead, oxidative surface activation techniques, such as ozone treatment were used for an increased shell stability.
FTO glass substrates (7 Ω/sq) were received from Pilkington, 3″ × 5 glass substrates were received from VWR and cut into 1 × 1 cm pieces. Gold and silver sputter targets (99.999% purity) were bought from Quorum Technologies Ltd and sputtered using a Quorum TS 150. 5-Carboxyfluorescein–alkyne (5-FAM) was purchased from Lumiprobe GmbH. All other chemicals used in this work were purchased from Sigma-Aldrich and used as received.
Synthesis of spherical Gd2O3 particles
The gadolinium oxide particles were prepared following the homogeneous precipitation method using urea . A Gd(NO3)3 solution (1 mL, 1 M) was placed in a 100 ml round bottom flask and was further diluted with 50 mL of water. Urea (2 g) was added to the flask and the mixture was stirred for 2 h at room temperature to form a homogeneous solution. Afterwards, reaction temperature was increased to 90 °C for 2 h to form a white milky dense solution. The particles were separated using a centrifuge at 11,000 rpm for 20 min, and washed three times with water, water–ethanol and ethanol. Finally, the particles were dried in an oven at 60 °C overnight. The particles were further annealed at 800 °C for 150 min (600 °C/h) to obtain Gd2O3 particles.
Synthesis of silica spheres (SiO2)
Silica particles were prepared using the Stöber method . In detail, ethanol (90.00 mL), deionized water (32.50 mL), and NH4OH solution (2.25 mL, 28%) were mixed together at 25 °C. Tetraethyl orthosilicate (TEOS, 7.75 mL, 35 mmol) was added under vigorous stirring and the dispersion was stirred for 2.5 h. Afterwards, the particles were collected by centrifugation (11,000 rpm, 15 min). The precipitated particles were redispersed in ethanol and water (1:1), followed by collection of the particles by centrifugation. This procedure was repeated once. Finally, the particles were dispersed in deionized water (15 mL).
Formation of azide functionalized silica spheres (SiO2–N3)
Azide functionalized silica spheres were prepared by surface treatment of the particles with 11-bromoundecyltrichlorosilane . The previously prepared SiO2 (200 mg, 3.33 mmol) sub-micrometer spheres were dispersed in a mixture of toluene (anhydrous, 50 mL) and dimethyl formamide (DMF, anhydrous, 5 mL), which was added to increase the dispersibility of the particles. Afterwards, the dispersion was heated up to 60 °C and 11-bromoundecyltrichlorosilane (422 µL, 1.44 mmol) was added rapidly. The reaction proceeded at 60 °C for additional 19 h. Particles were separated by centrifugation and washed at least four times with toluene. The final particles were dried for 12 h at ambient conditions. Afterwards, the bromine functionalized particles were dispersed in DMF (5 mL) and NaN3 (200 mg, 3.07 mmol) was added. The reaction was stirred for 40 h at 25 °C. Collection of the particles was performed with a centrifuge (4400 rpm, 15 min), followed by five washing steps. Finally, the particles were dried for 12 h at 25 °C.
Formation of thiolated silica spheres (SiO2–SH)
Thiol functionalized silica spheres were prepared according to Claesson et al. . In this procedure, 3-mercaptopropyl trimethoxysilane (MPTMS, 1.5 mL, 7.16 mmol) was added to an aqueous dispersion (70 mg/mL) of silica spheres. The reaction proceeded for 45 min at ambient conditions. Afterwards, the solution was heated to 80 °C for 60 min. The particles were collected via centrifugation (9 000 rpm, 10 min) and washed three times with ethanol. Finally, the particles were dispersed in ethanol (40 mL).
FTO and glass substrates were washed for 15 min each with acetone, deionized water with 2% Helmanex III soap, and isopropyl alcohol in an ultrasonic bath kept at 50 °C. Before spin coating, the FTOs were treated with oxygen plasma for 15 min and the glass substrates were treated oxidatively in an UV–ozone cleaner (Dinier©, ELG 100 s) to remove any remaining organic impurities and to increase the surface wettability. All substrates were used directly after activation.
Preparation of Janus-type Gd2O3@Ag and Gd2O3@Au particles
The prepared Gd2O3 particles (50 mg) were placed in an argon flushed vial equipped with a stirring bar and transferred to a nitrogen filled glovebox. Isopropanol (anhydrous, 600 µL) was added to form a homogeneous white dispersion stirred for 2 h. FTO substrates were also transferred to the glovebox and each was coated with Gd2O3–isopropanol solution (50 µL). Spin coating of substrates was performed with an acceleration rate of 800 rpm/s for 45 s at 1000 and 3000 rpm, respectively. The coated substrates were placed on a hot-plate at 100 °C to dry and ensure removal of solvent. Coated substrates were placed inside a sputtering machine. Silver and gold were sputtered onto the particles layer at a discharge current of 20 mA at 20 mbar for 20 and 40 s, respectively.
Preparation of Janus-type SiO2–N3@Au and SiO2–SH@Au particles
SiO2–N3 or SiO2–SH dispersion (10 µL, 70 mg mL−1) was transferred onto an activated glass slide. The dispersion was allowed to cover the substrate completely and dried using a spin-coating technique. The dried sample was placed in the UV chamber for further 30 min and gold was sputtered onto the substrates (20 mA, 30 s). Finally, the particles were collected in ethanol (4 mL) by sonification.
Covalent attachment of 5-FAM to SiO2–N3@Au particles
CuSO4 (12.5 mg, 0.07 mmol), l-histidine (19.5 mg, 0.13 mmol), and sodium ascorbate (49 mg, 0.25 mmol) were each dissolved in water (250 µL). Afterwards, CuSO4, a 5-FAM-alkyne solution (100 µL, 5 mM), histidine and sodium ascorbate were mixed subsequently under stirring in a 10 mL reaction vessel. Finally, the aqueous dispersion of SiO2–N3@Au particles (1 mL, ~ 7 mg mL−1) was added and the reaction was allowed to proceed for 4 h. The final particles were collected via centrifugation (1600 rpm, 15 min) and washed five times. Finally, the particles were dried for 14 h under ambient conditions.
All measurements were performed under ambient conditions and in neutral pH. X-ray diffraction (XRD) analysis of Gd2O3 particles was carried out on an STOE-STADI MP diffractometer equipped with a Cu (R = 0.15406 nm) source and operating in transmission mode. A scan rate of 0.05°/s was applied to record the pattern in the 2θ range of 10°–80°. Scanning electron micrographs were performed on an FEI Nova Nano SEM 430. Energy-dispersive X-ray spectroscopy was performed on an Apollo X EDAX. (Working distance 5 mm; entry angle 35°). X-ray photoelectron spectroscopy (XPS) was performed on an ESCA M-Probe (Surface Science Instruments) using Al-Kα-radiation (1486.6 eV). TEM measurements were carried out using a ZEISS LEO 902 microscope operating at 120 kV with LaB6 cathode in a bright field mode. The samples were deposited onto a carbon coated copper grid. The mean diameter was statistically determined from a varying number of particles from bright field micrographs. Dynamic light scattering (DLS) and ζ-potential measurements were performed with a Malvern Instruments Zetasizer Nano ZS (operating wavelength: 633 nm). All values and standard deviations presented here were calculated out of five measurements. Measurements were performed in polystyrene cuvettes. FT-IR spectra were collected using a Perkin Elmer Spectrum 400. Powder-like samples were measured in the range of 4000–400 cm−1. For Raman spectroscopy, a Horiba Jobin–Yvon spectrometer in triple subtractive mode equipped with a liquid nitrogen-cooled CCD detector, 1800 gr/mm gratings, and a laser wavelength of 532 nm was used. The incident beam angle was 45° concerning the a–b plane of the sample. Before the measurement, the powder-like sample was placed between two previously cleaned microscopic glass slides.
Results and discussion
Particle preparation and functionalization
The electron microscope images in Fig. 2b illustrate the size and morphology of the Gd(OH)CO3∙H2O particles before thermal treatment. As synthesized particles were spherical in shape, nearly, monodisperse and homogeneously distributed with average size of 100–120 nm, fitting the demand of biological application. Furthermore, the particles’ uniform surface is preferred for targeting ligands’ conjugation and imaging probes . It is noteworthy that the particles did not show any changes in size or morphology after calcination, as clearly presented in Fig. 2c. Even for a rapid heating rate of 10 °C/min, the particles maintained their regular morphology in contrast to the previous work of Di et al., reporting an optimal heating rate of 2 °C/min to prevent shrinkage of the particles and maintaining their original morphology .
As presented in Fig. 3a, a clear signal was visible at 2102 cm−1, related to the successful attachment of the azide onto the particles surface [16, 35]. DLS analysis proves the formation of particles with an average hydrodynamic diameter of 229 (4) nm and an average ζ-potential of – 19 (2) mV, where the replacement of negatively charged hydroxyl functions by azide groups resulted in the observed shift towards positive values of the ζ-potential.
For obtaining thiol functionalized silica particles, potentially usable for thiol-ene coupling, previously prepared silica particles were surface functionalized with MPTMS . Raman spectroscopy proved a successful attachment of the thiol function on the particles surface (Fig. 3b). The bands related to the thiol group are measured at 2571 cm−1 (marked in red, S–H bond stretching), 1261 cm−1 (wag vibration of the S–CH2 group), and 650 cm−1 (C–S bond stretching) , while C–H bands were observed at 2928 cm−1 (antisymmetric C–H stretching of the CH2 groups) and 2894 cm−1 (symmetric C–H stretching of the CH2 groups), as well as at 1431 cm−1 (CH2 deformation). This indicates that non-oxidized thiol groups are present on the surface. Therefore, an immobilization of target molecules via thiol-ene reactions should be possible. The average hydrodynamic diameter of SiO2–SH particles was measured as 267 (3) nm by DLS analysis, whereas the obtained ζ-potential was – 45 (1) mV. As in case of silica particles, this negative value indicates a long-term dispersability and a negative surface charge consisting of deprotonated thiol groups.
Janus particle formation
For controllable asymmetric functionalization, previously described particles were immobilized on carrier substrates using spin-coating technique. The substrates were treated either with oxygen plasma or UV–ozone prior to particle deposition, providing a good solvent wettability and easy removal of organic residues from the substrate surface. The coated substrates were dried subsequently to remove any remaining solvent. A thin layer (< 10 nm) of silver and gold was deposited onto the surface of the NPs via magnetron sputtering at various discharge currents and times, depending on the material. The resulting coated particles were then released from the supporting substrate and redispersed in an aqueous solution by immersing the substrate under sonication for a few seconds.
In fact, all samples showed long-term stability even after removal from the substrate surface, as confirmed by SEM measured for the same particle dispersions after weeks (Figure S2). It can be clearly seen that the sputtered metallic clusters adhere strongly to the Gd2O3 surface and the dispersion of particles using ultrasonic bath did not deform the spheres or detached metallic NPs. Moreover, it has been reported that the deposition of noble metals on metal-oxide surfaces needs an adhesion layer such as Ti/W alloy or Ni, to obtain an intimate and long lasting contact of the film [28, 29]. However, it should be noted that asymmetrical capping of the presented particles with Ag and Au was achieved without any additional interlayer support.
XPS analysis of Janus-type Gd2O3 particles
The composition and interaction between noble metals and ceramic host particles (Gd2O3) were studied in detail by XPS, whereby high-resolution spectra were collected for detailed investigations of the interface between plasmonic surface and oxidic host particle. Charge correction was carried out in all spectra considering the charge shift of the C 1 s peak of adventitious carbon. An XPS survey spectrum of bare Gd2O3 NPs is presented in Figure S3, demonstrating a peak at 136.3 eV which is related to the binding energy of Gd 4d, whereas the presence of adventitious carbon from atmospheric exposure after spin-coating procedure is confirmed by the C 1 s peak at 284.81 eV. The O 1 s surface peak at 530.31 eV is dedicated to Gd2O3 and in good agreement with reported data of O 1 s in Gd2O3 at 530.6 eV .
Further functionalization of silica Janus-type particles
As already reported by our group , click chemistry has a high potential for the bioconjugation of NPs. To investigate the applicability of SiO2–N3@Au particles presented here, a model molecule (5-Carboxyfluorescein, 5-FAM) was selectively attached to the azide functionalized side of the particles via a copper catalyzed click reaction .
Plasmonic silver and gold NPs were successfully immobilized on phase pure Gd2O3 and functionalized silica spheres, leading to the formation of non-centrosymmetric dispersable systems. In contrast to literature reports, no adhesive interlayer was needed to guarantee an intimate contact at the NP–metal interface, still present after weeks, as proven by SEM and TEM. UV–Vis spectroscopy revealed a plasmonic activity for Gd2O3@Ag, Gd2O3@Au, and SiO2–N3@Au. In case of Gd2O3@Ag and Gd2O3@Au, the red shift of the absorption bands was explained by shell effects, such as variations in roughness and the presence of nanoclusters. In addition, XPS analysis proved the metallic character of the silver and gold NPs, only a few oxidized AgO species were observed, which could be attributed to the exposure of the sputtered sample to aerated conditions. Further functionalization of SiO2–N3@Au with a model molecule (5-FAM) via click chemistry was successfully performed and proven by UV–Vis and FT-IR spectroscopy. Our results demonstrate that this synthesis route avoids the use of expensive reagents and solvents and is capable of generating functional layers on sub-micrometer sized particles without additional adjustments. Furthermore, we prove the applicability of the presented approach to a variety of crystalline and amorphous systems, providing new possibilities in material and interface engineering.
The authors would like to thank the University of Cologne (Excellence Program “Quantum Matter and Materials”), the “Deutsche Forschungsgemeinschaft” (DFG) and the “Deutscher Akademischer Austauschdienst” (DAAD) for financial support. S. Öz gratefully acknowledges the financial support provided by Merck KGaA (Darmstadt). In addition, we are thankful to Dipl.-Phys. Raphael German for Raman spectroscopy measurements, Mrs. Nurgül Tosun and Dr. Stefan Roitsch for SEM and TEM measurements.
Compliance with ethical standards
Conflict of interest
The authors declare no competing financial interest.
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