Growing Gold Nanostars on 3D Hydrogel Surfaces

Abstract

Nanocomposites comprising hydrogels and plasmonic nanoparticles are attractive materials for tissue engineering, bioimaging, and biosensing. These materials are usually fabricated by adding colloidal nanoparticles to the uncured polymer mixture and thus require time-consuming presynthesis, purification, and ligand-exchange steps. Herein, we introduce approaches for rapid synthesis of gold nanostars (AuNSt) in situ on hydrogel substrates, including those with complex three-dimensional (3D) features. These methods enable selective AuNSt growth at the surface of the substrate, and the growth conditions can be tuned to tailor the nanoparticle size and density (coverage). We additionally demonstrate proof-of-concept applications of these nanocomposites for SERS sensing and imaging. High surface coverage with AuNSt enabled 1-2 orders of magnitude higher SERS signals compared to plasmonic hydrogels loaded with premade colloids. Importantly, AuNSt can be prepared without the addition of any potentially cytotoxic surfactants, thereby ensuring a high biocompatibility. Overall, in situ growth becomes a versatile and straightforward approach for the fabrication of plasmonic biomaterials.

Figure 1

(A) Schematic illustration of the growth of gold nanostars (AuNSt) from colloidal Au@citrate seeds in the presence of laurylsulfobetaine (LSB) and triton X-100 (TX) as surfactants. The chemical structure of the ligands and digital photographs of the final substrates are shown below the scheme. TEM images of AuNSt@LSB (B) and AuNSt@TX (C), collected after dissolving GelMA. SEM images showing AuNSt at the hydrogel surface for AuNSt@LSB (D, F) and AuNSt@TX (E, G). Scale bars of insets: 100 nm. Additional SEM images are provided in Figures S2–S4, and size distributions are plotted in Figure S5. SEM images of the substrate after incubation in a growth solution containing LSB (H) and TX (I), with no seeding step (control experiments).

Figure 2

(A) Schematic view of the process for in situ single-step growth for gold nanostars on gelatin methacryloyl. (B) SEM image of the products on the gel surface, and (inset) TEM image of particles collected from the dissolved gel. (C) TEM image of the particles formed in the growth solution (as a result of secondary nucleation, not grown on the substrate; Ø = 26 ± 11 nm for the supernatant and Ø tip-to-tip = 41 ± 18 nm for in situ products, respectively; n = 150). (D) Size distribution of the products in the growth solution vs those formed on the substrate. (E) Scheme describing the synthesis of gold nanostars in a preloaded fashion, where nanoparticles form specifically on the substrate, rather than both on the substrate and in solution. (F) SEM image of nanostars prepared using the preloaded approach, and (inset) TEM image of particles collected after dissolving the gel. Additional SEM images are provided in Figures S9, S10, and S12, and size distributions are plotted in Figure S11.

Figure 3

(A) Schematic representation of the reaction between gelatin and methacrylic anhydride to produce gelatin methacryloyl (GelMA) and subsequent GelMA cross-linking. (B, C) Raman scattering spectra showing changes in the characteristic GelMA peaks after incubation of the gel with HAuCl₄ for 24 h. SEM images of gold nanostars formed on 10% w/w gelatin (nonmethacrylated) by single-step (D) and preloaded (E) in situ growth (insets: digital photographs of gelatin substrates after AuNSt growth).

Figure 4

(A) Schematic showing the procedure for preparing hydrogels with 4-mercaptobenzoic acid labeled gold nanostars prepared in colloidal suspension. (B) SEM image showing the surface of a hydrogel mixed with a AuNSt colloid (=0.5 mM; Abs₄₀₀ = 1.2). Few nanostars can be observed in the SEM images, indicated by blue circles. Surface-enhanced Raman scattering (SERS) depth slice maps (1–10 mW, 0.01–0.05 s integration; full details provided in the Supporting Information) showing the localization of the SERS signal corresponding to the characteristic peak of 4-mercaptobenzoic acid (4-MBA) at 1082 cm^–1, for substrates prepared using the different approaches: with colloidal nanostars (C), or seeded in situ growth using laruylsulfobetaine (LSB) (D) and Triton X-100 (TX) (E) as surfactants, single-step in situ growth (F), and preloaded in situ growth (G). SEM images showing the substrates in a cross-section profile for seeded in situ growth with LSB (H) and TX (I) as capping ligands, single-step in situ growth (J), and preloaded in situ growth (K) substrates. The visibility of the sample surface differs from sample to sample depending on the angle of the surface relative to the detector upon drying. Dashed lines were added as a guide to the eye, indicating the division between the internal part of the gel and the surface coated with nanostars. Low-magnification SEM images of the dried cross sections are shown in Figure S19.

Figure 5

(A) Representative surface-enhanced Raman scattering (SERS) spectra for substrates prepared with colloidal nanostars (“colloids”), the seed-mediated in situ growth approaches with laurylsulfobetaine and Triton X-100 (“LSB” and “TX,” respectively), single-step in situ growth, and preloaded in situ growth. (B) Comparison of the uniformity of the intensity of the peak corresponding to Raman reporter molecule 4-mercaptobenzoic acid (4-MBA) for the substrates made by each method (n = 5 for each). (C) Comparison of the percent error in the 4-MBA SERS signal (spectra taken at 7.1 mW (power at surface), 3 s accumulation, 50× long working distance objective, NA = 0.5). (D) Normalized UV–visible extinction spectra of the nanocomposites.

Figure 6

(A) Digital photograph showing an entire 3D scaffold with irregular surface features coated with gold nanostars (AuNSt), which provide a dark blue color. (B–D) SEM images of a cross-section of the scaffold. Cross-section areas are indicated by dashed lines. A specific region of interest at the cross-section edge common to all images is indicated by yellow dashed lines. (E) SERS x,y-map of a scaffold functionalized with 4-mercaptobenzoic acid (10× objective, NA = 0.25, 43.6 mW, 0.01 s accumulation). (F) Fluorescence confocal microscopy images of green fluorescence protein (GFP)-expressing MDA-MB-231 breast cancer cells seeded on a AuNSt scaffold functionalized with collagen/fibronectin. (G) Representative SERS spectra off and on the cells (differences indicated by arrows). (H) SERS map of seeded cells, based on the intensity at 951 cm^–1. Additional fluorescence microscopy images and cell viability evaluation in 2D and 3D are shown in Figures S21 and S22, and additional SERS characterization is provided in Figures S23 and S24.