Plasmonic nanoparticles track dynamic behavior of molecules in live cells

Molecular interactions govern a myriad of normal and pathologic human processes. Developing methods that allow us to see how biomolecules behave in live cells will lead to better understanding of the cellular and molecular underpinnings of devastating diseases such as cancer. To date, fluorescence-resonance energy transfer (FRET) has been the fundamental tool in imaging and understanding inter- and intramolecular interactions.^1,2 However, FRET has a number of shortcomings that limit its application to a broader range of biomedical problems, including photobleaching and low efficiency. Moreover, the technique is sensitive only to molecular interactions that occur at short distances (typically ~5nm), which requires painstaking chemical labeling protocols to precisely position the donor and acceptor fluorophors within the molecules of interest.

Distance-dependent coupling of plasmon resonances between closely spaced metal nanoparticles offers an attractive alternative for imaging molecular interactions. The advantages of plasmon coupling include a dramatic nonlinear increase in scattering cross-section per interacting particle,^3,4 alterations in plasmon resonance frequency (color change),⁴ and depolarization of linearly polarized light.⁵ The distances over which coupling is significant can be as large as three times the particle radius, thus extending the range of detectable protein interaction distance more than an order of magnitude relative to FRET.^6,7 Consequently, interactions can be imaged across cellular membranes. They can also be detected between two biomarkers that may be separated by one or more intermediate or adapter proteins linking them together. Plasmonic nanoparticles offer other advantages. They have optical cross-sections that are many times larger than fluorescent dyes, green fluorescent protein, or even quantum dots.^8,9 They are chemically inert.¹⁰ They have stable signal intensity because of a lack of photobleaching or blinking effects.⁹ Finally, they are amenable to surface modification and functionalization strategies that allow synthesis of multifunctional nanoparticles.^11–13 The tremendous potential of plasmonic nanoparticles as optically interrogatable biological labels has already been recognized and has led to a variety of novel applications in bioanalytical chemistry with unprecedented sensitivity.^8,9,14–20

The work we describe here expands and generalizes the application of nanoparticle plasmon-resonance coupling (NPRC) to monitor the dynamic behavior of large protein complexes in living cells.²¹ This has important implications, as there is currently a dearth of physical methods for monitoring the behavior of large molecular complexes with high spatial and temporal resolution in living cells. We observed the trafficking mechanisms of the epidermal growth-factor receptor (EGFR), a key receptor tyrosine kinase that controls fundamental cellular processes such as DNA replication and division.²² EGFR is also an important cancer biomarker.^23,24

We labeled growth-factor receptors with molecular-specific gold nanoparticles and showed that major stages of receptor regulation are associated with distinct color changes of plasmonic nanoparticles (see Figure 1). This relationship allowed us to monitor nanometer-scale receptor behavior in living cells. We obtained hyperspectral-imaging (across a large spectral range) and electron-microscopy data from a series of temperature points corresponding to arrested EGFR regulatory states. We employed detailed electrodynamic modeling to verify the physical basis of the optical process under observation. Statistical analysis of the hyperspectral data made it possible to assign a false-color map to RGB (red, green, blue) images to produce a real-time map of the spatial distribution of EGFR regulatory stages in live cells. This combination of nanoparticle plasmonic coupling with statistical image analysis could be applied in the near term in robust high-throughput assays for many biological processes that involve molecular assemblies. The availability of in vivo optical-imaging methods also provides a potential translational route for these assays to the monitoring of molecular therapy in vivo.

Figure 1. Dark-field image of a live cell ∼15min after labeling with anti-epidermal growth-factor-receptor (EGFR) gold nanoparticles (left). Over time, the labeling pattern undergoes progressive color change from green (at 15min) to yellow (~30min), and finally to orange-red (>50min), as shown in the middle column (the images display the region that is highlighted by the white box on the left). The color changes are well correlated with the trafficking dynamics of EGFR, which include receptor-molecule dimerization (pairing) and aggregation in the plasma membrane, followed by endocytosis (uptake) into early endosomes (intracellular compartments) and, finally, formation of late endosomes. The transmission-electron-microscope (TEM) pictures (right) confirm changes in the nanoscale organization of labeled receptor molecules. Optical images were acquired using transmitted dark-field illumination and a long-working-distance 63×, 0.75NA (numerical aperture) objective. The scale bar is ~10μm in the left panel and ~200nm in the TEM images (right).

A primary strength of NPRC as a biosensing tool derives from the complex optical behavior of plasmonic nanoparticle assemblies. The dramatic changes in optical properties that correlate with nanometer-scale alterations in the organization of labeled biomolecules facilitate statistical analysis of the processes under observation. Further understanding of NPRC will be vital for its development as a quantitative biosensing tool. We are continuing to study issues such as heterogeneity of particle shape and the more nuanced effects of nanoparticle aggregate morphology on spectral characteristics (such as plasmon peak width), which promise to reveal a wealth of further information.

We gratefully acknowledge support from National Cancer Institute grant R01 CA103830 BRP.