Optimization of immunolabeled plasmonic nanoparticles for cell surface receptor analysis.

Noble metal nanoparticles hold great potential as optical contrast agents due to a unique feature, known as the plasmon resonance, which produces enhanced scattering and absorption at specific frequencies. The plasmon resonance also provides a spectral tunability that is not often found in organic fluorophores or other labeling methods. The ability to functionalize these nanoparticles with antibodies has led to their development as contrast agents for molecular optical imaging. In this review article, we present methods for optimizing the spectral agility of these labels. We discuss synthesis of gold nanorods, a plasmonic nanoparticle in which the plasmonic resonance can be tuned during synthesis to provide imaging within the spectral window commonly utilized in biomedical applications. We describe recent advances in our group to functionalize gold and silver nanoparticles using distinct antibodies, including EGFR, HER-2 and IGF-1, selected for their relevance to tumor imaging. Finally, we present characterization of these nanoparticle labels to verify their spectral properties and molecular specificity.

Polarization mapping of nanoparticle plasmonic coupling.

We propose the use of polarization mapping as a tool to better separate the effects of plasmonic coupling from the local refractive index for molecular imaging and biosensing using gold nanoparticles. Polarization mapping allows identification of the orthogonal excitation mode when the particle dimer orientation is unknown, as may be the case when using plasmonic nanoparticles for cell labeling. This information can be used to sense relative changes in the dielectric environment, or for absolute dielectric sensing with additional a priori interparticle distance information. First, the theoretical scattering by nanoparticle pairs is modeled under parallel and orthogonal polarization orientations and increasing interparticle separation. Second, polarization mapping of substrate bound nanoparticles using dark-field microspectroscopy is investigated as a method to isolate the individual plasmonic coupling modes associated with a pair of nanoparticles without reorientation of the sample. The results of this study provide useful insight toward potential avenues for monitoring distances using plasmonic nanoparticles and sensing the local refractive index using nanoparticle pairs when the pair orientation is not known, as may be the case when using nanoparticles for cell receptor labeling.

Feasibility study of brain tumor delineation using immunolabeled gold nanorods.

Effective treatment of patients with malignant brain tumors requires surgical resection of a high percentage of the bulk tumor. Surgeons require a method that enables delineation of tumor margins, which are not visually distinct by eye. In this study, the feasibility of using gold nanorods (GNRs) for this purpose is evaluated. Anti-Epidermal Growth Factor Receptor (anti-EGFR) conjugated GNRs are used to label human xenograft glioblastoma multiforme (GBM) tumors embedded within slices of brain tissues from healthy nude mice. The anti-EGFR GNRs exhibit enhanced absorption at red to near-infrared wavelengths, often referred to as the tissue optical window, where absorption from blood is minimal. To enable definition of molecular specificity and spatial accuracy of the label, the GNR absorption is compared with GFP fluorescence which is expressed by the GBM cells used here. This work demonstrates a simple but highly translational technique to classify normal and malignant brain tissue regions in open surgery applications using immunolabeled GNR contrast agents.

Multispectral nanoparticle contrast agents for true-color spectroscopic optical coherence tomography.

We have recently developed a novel dual window scheme for processing spectroscopic OCT images to provide spatially resolved true color imaging of chromophores in scattering samples. Here we apply this method to measure the extinction spectra of plasmonic nanoparticles at various concentrations for potential in vivo applications. We experimentally demonstrate sub-nanomolar sensitivity in the measurement of nanoparticle concentrations, and show that colorimetric imaging with multiple species of nanoparticles produces enhanced contrast for spectroscopic OCT in both tissue phantom and cell studies.

Monitoring of receptor dimerization using plasmonic coupling of gold nanoparticles.

The dimerization of receptors on the cell membrane is an important step in the activation of cell signaling pathways. Several methods exist for observing receptor dimerization, including coimmunoprecipitation, chemical cross-linking, and fluorescence resonance energy transfer (FRET). These techniques are limited in that only FRET is appropriate for live cells, but even that method suffers from photobleaching and bleed-through effects. In this study, we implement an alternative method for the targeting of HER-2 homodimer formation based on the plasmonic coupling of gold nanoparticles functionalized with HER-2 Ab. In the presented studies, SK-BR-3 cells, known to overexpress HER-2, are labeled with these nanoparticles and receptor colocalization is observed using plasmonic coupling. HER-2 targeted nanoparticles bound to these cells exhibit a peak resonance that is significantly red-shifted relative to those bound to similar receptors on A549 cells, which have significantly lower levels of HER-2 expression. This significant red shift indicates plasmonic coupling is occurring and points to a new avenue for assessing dimerization by monitoring their colocalization. To determine that dimerization is occurring, the refractive index of the nanoenvironment of the labels is assessed using a theoretical analysis based on the Mie coated sphere model. The results indicate scattering by single, isolated nanoparticles for the low HER-2 expressing A549 cell line, but the scattering observed for the HER-2 overexpressing SK-BR-3 cell line may only be explained by plasmonic-coupling of proximal nanoparticle pairs. To validate the conformation of nanoparticles bound to HER-2 receptors undergoing dimerization, discrete dipole approximation (DDA) models are used to assess spectra of scattering by coupled nanoparticles. Comparison of the experimental results with theoretical models indicates that NP dimers are formed for the labeling of SK-BR-3 cells, suggesting that receptor dimerization has been observed.

Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles.

This work presents simultaneous imaging and detection of three different cell receptors using three types of plasmonic nanoparticles (NPs). The size, shape, and composition-dependent scattering profiles of these NPs allow for a system of multiple distinct molecular markers using a single optical source. With this goal in mind, tags consisting of anti-epidermal growth factor receptor gold nanorods, anti-insulin-like growth factor 1-R silver nanospheres, and human epidermal growth factor receptor 2Ab gold nanospheres were developed to monitor the expression of receptors commonly overexpressed by cancer cells. These labels were chosen because they scatter strongly in distinct spectral windows. A hyperspectral darkfield microspectroscopy system was developed to record the scattering spectra of cells labeled with these molecular tags. Simultaneous monitoring of multiple tags may lead to applications such as profiling of cell line immunophenotype and investigation of receptor signaling pathways. Single, dual, and triple tag experiments were performed to analyze NP tag specificity as well as their interactions. Distinct resonance peaks were observed in these studies, showing the ability to characterize cell lines using conjugated NPs. However, interpreting shifts in these peaks due to changes in a cellular dielectric environment may be complicated by plasmon coupling between NPs bound to proximal receptors and other coupling mechanisms due to the receptors themselves.