Cellular imaging by targeted assembly of hot-spot SERS and photoacoustic nanoprobes using split-fluorescent protein scaffolds

Abstract

The in cellulo assembly of plasmonic nanomaterials into photo-responsive probes is of great interest for many bioimaging and nanophotonic applications but remains challenging with traditional nucleic acid scaffolds-based bottom-up methods. Here, we address this quandary using split-fluorescent protein (FP) fragments as molecular glue and switchable Raman reporters to assemble gold or silver plasmonic nanoparticles (NPs) into photonic clusters directly in live cells. When targeted to diffusing surface biomarkers in cancer cells, the NPs self-assemble into surface-enhanced Raman-scattering (SERS) nanoclusters having hot spots homogenously seeded by the reconstruction of full-length FPs. Within plasmonic hot spots, autocatalytic activation of the FP chromophore and near-field amplification of its Raman fingerprints enable selective and sensitive SERS imaging of targeted cells. This FP-driven assembly of metal colloids also yields enhanced photoacoustic signals, allowing the hybrid FP/NP nanoclusters to serve as contrast agents for multimodal SERS and photoacoustic microscopy with single-cell sensitivity.

Fig. 1

Characterization of AuNPs functionalized with split-fluorescent protein fragments. a Schematic of surface modification on AuNPs by sGFP and M3 peptide fragments and formation of SERS active hot spot through self-assembly and GFP complementation. b Comparison of the absorption spectra of bare AuNPs (solid black), AuNPs coated with M3 peptides (M3-AuNPs, solid red), and AuNPs coated with sGFP (sGFP-AuNPs, dash blue). Inset: size exclusion chromatography of M3-AuNPs. c Immuno-dot blot characterization of the presence of full-length GFP or sGFP at the surface of AuNPs. Scale bars: 0.5 cm d TEM images of monodispersed AuNPs coated with M3 peptides or sGFP. Scale bar: 200 nm. e DLS size distributions of bare AuNPs, M3-AuNPs and sGFP-AuNPs, coated with different size PEGs. The hydrodynamic diameter ( ± s.d.) was determined for n = 30 repetitive measurements for each sample

Fig. 2

Formation of AuNP clusters by the assembly of FP fragments. a Agarose gel electrophoresis of sGFP-AuNPs, M3-AuNPs and unpurified sGFP-AuNPs + M3-AuNPs mixture and typical TEM images of clusters assembled with 40/40 nm AuNPs (top), 40/20 nm AuNPs (middle), and 40/10 nm AuNPs (bottom). White rectangles indicate the position of cluster bands in gels. Scale bars: 20 nm. b TEM images of clusters formed by the assembly of M3-AuNPs with sGFP-AuNPs, sYFP-AuNPs, or sCFP-AuNPs. Scale bars: 200 nm. c Normalized AuNP cluster size distribution and fit by a power law distribution model. d Size distribution of n = 314 nanogaps formed by the assembly of FP fragments between AuNPs in clusters. The distribution is Gaussian and centered at 2.1 ± 0.5 nm (mean ± s.d.). Inset: expected orientation of complemented GFP at nanogaps. e TEM images of clusters formed by the assembly of sGFP-gold nanorods with different sizes of M3-AuNPs. Scale bars: 20 nm

Fig. 3

SERS spectra of complemented sGFP and assembled AuNP clusters. a SERS spectra of full-length GFP (blue), M3 peptide-complemented split-GFP (red), and non-complemented sGFP fragment (green) on 5 nm silver island substrates. b Liquid SERS spectra of assembled AuNP clusters at pH 8.0 (left) and pH 6.0 (right)

Fig. 4

Cell targeting and plasma membrane clustering of biotinylated AuNPs. a Schematic of biotinylated M3-AuNPs and sGFP-AuNPs targeted to avidin fusion proteins at the plasma membrane of cells and their assembly into SERS active clusters. Not to scale. b Bright field (left), single-frame TIRF microscopy image (middle) and maximum intensity projection TIRF image from multiple frames (∑I_max, right) of biotinylated M3-AuNPs bound to GPI-avidin fusion proteins and diffusing at the plasma membrane of live HeLa cells. Scale bar: 7 μm. c Scanning electron microscope images of U2OS cells co-expressing the transmembrane and the GPI-anchored avidin fusion proteins and targeted with biotinylated sGFP-AuNPs alone (top panel), biotinylated M3-AuNPs alone (bottom panel) or both biotinylated sGFP-AuNPs and M3-AuNPs simultaneously (middle panel). White arrows point towards endocytic membrane structures, blue arrowheads point towards AuNPs monomers and red arrowheads point towards some of the AuNP nanoclusters presented in insets. The plus and minus signs identify the avidin-expressing and non-expressing cells, respectively. Scale bars: 2 μm (left panels), 200 nm (insets of left panels), 1 μm (right panels), and 100 nm (insets of right panels). d Cluster size distributions of AuNPs on targeted U2OS cells

Fig. 5

Targeted SERS imaging of cells with split-FP assembled metal nanoclusters. a SERS microscopy images of fixed cells at the GFP chromophore 1527 cm⁻¹ imidazolinone/exocyclic C=C Raman mode and corresponding SERS spectra on cells expressing the avidin biomarkers and targeted by biotinylated M3-AuNPs and sGFP-AuNPs separately or simultaneously. Colored arrows in images point toward single pixels whose SERS spectra are represented in matching colors. The three typical GFP chromophore vibrational modes are indicated by dash lines on spectra. b SERS microscopy image of live cells co-targeted by biotinylated M3-AuNPs and sGFP-AuNPs and reconstructed at a 1535 ± 15 cm⁻¹ spectral window. The deconvolved SERS spectrum corresponds to one pixel in the cell image as indicated by the arrow. c SERS image of live cells co-targeted by biotinylated M3-AgNPs and sGFP-AgNPs and reconstructed at a 1550 ± 15 cm⁻¹ spectral window. The SERS spectrum corresponds to the individual pixel indicated by the arrow in the cell image. d SERS image of live cells in hypotonic buffer after co-targeting of biotinylated M3-AuNPs and sGFP-AuNPs and reconstruction at a 1550 ± 15 cm⁻¹. The SERS spectrum corresponds to one pixel in the cell image as indicated by the arrow. All scale bars: 10 µm

Fig. 6

SERS imaging of cancer cells with folate-functionalized AuNPs. a Wide-field fluorescence imaging of folate receptor expression in human carcinoma KB cells (left) and human primary dermal fibroblasts (right). Scale bars: 20 µm. b Reflection image, SERS microscopy image reconstructed at a 1565 ± 15 cm⁻¹ spectral window and SERS spectrum of individual pixels (colored arrows) for live carcinoma KB cells co-targeted by folate-functionalized M3-AuNPs and sGFP-AuNPs. Scale bars: 20 µm (left) and 10 µm (right). c Reflection image, SERS microscopy image reconstructed at a 1565 ± 15 cm⁻¹ spectral window and SERS spectrum of individual pixels (colored arrows) for a live primary dermal fibroblast co-targeted by folate-functionalized M3-AuNPs and sGFP-AuNPs. Scale bars: 20 µm (left) and 10 µm (right)

Fig. 7

Photoacoustic imaging of in situ assembled split-FP AuNP clusters on cells. a Photoacoustic microscopy images of individual U2OS cells among a 100% confluent field after targeted clustering of biotin-M3-AuNPs and biotin-sGFP-AuNPs on cells that transiently express plasma membrane avidin biomarkers. Scale bars: 50 µm (left) and 20 µm (right). b Photoacoustic signal amplitudes from similar fields of U2OS cells targeted with both biotin-M3-AuNPs and biotin-sGFP-AuNPs (split-FP clustered) or with biotin-sGFP-AuNPs only (non-clustered). The mean photoacoustic signal amplitudes (±s.d.) were determined for n = 6 fields of views totaling 3 mm² of cells at 100% confluence for each condition. ***p < 0.01, t test. c Photoacoustic signal amplitudes from targeted U2OS cells at increasing laser excitation energy. The mean photoacoustic signal amplitudes (±s.d.) were determined for n = 4 fields of views with cells at 100% confluence for each condition. Lines represent linear regression fit of the data. d Photoacoustic microscopy images of carcinoma KB cells targeted with both folate-sGFP-AuNPs and folate-M3-AuNPs (clustered AuNPs, left), of carcinoma KB cells targeted with folate-sGFP-AuNPs only (non-clustered AuNPs, middle) and of primary dermal fibroblasts targeted with both folate-sGFP-AuNPs and folate-M3-AuNPs (clustered AuNPs, right). Scale bars: 50 µm