Plasmonic flow cytometry by immunolabeled nanorods

Abstract

Fluorescence-based flow cytometry measures multiple cellular characteristics, including levels of receptor expression, by assessing the fluorescence intensity from a population of cells whose cell surface receptors are bound by a fluorescently labeled antibody or ligand for that receptor. Functionalized noble metal nanoparticles provide a complementary method of receptor labeling based on plasmonics for population analysis by flow cytometry. The potential benefits of using plasmonic nanoparticles to label cell surface receptors in flow cytometry include scattering intensity from a single particle that is equivalent to fluorescence intensity of 10 5 fluorescein molecules, biocompatibility and low cytotoxicity, and nonquenching optical properties. The large spectral tunability of nanorods also provides convenient access to plasmonic markers with peak surface plasmon resonances ranging from 600 to 2,200 nm, unlike gold nanosphere markers that are limited to visible wavelengths. Gold nanorod-based plasmonic flow cytometry is demonstrated herein by comparing the scattering of cells bound to antiepidermal growth factor receptor (EGFR)-conjugated nanorods to the emission of cells bound to anti-EGFR-conjugated fluorescent labels. EGFR-expressing cells exhibited a statistically significant six-fold increase in scattering when labeled with anti-EGFRconjugated nanorods compared with labeling with IgG1-conjugated nanorods. Large scattering intensities were observed despite using a 1,000-fold lower concentration of nanorod-conjugated antibody relative to the fluorescently labeled antibody. ' 2010International Society for Advancement of Cytometry Key terms flow cytometry; nanorod; plasmonic markers; epidermal growth factor receptor NOBLE metal nanoparticles exhibit unique optical properties that can be exploited for a variety of applications. These properties arise from the interaction of an incident electromagnetic (EM) field with free conduction band electrons in the nanoparticle. The conduction electrons are polarized relative to the heavier positive core ions because of the exciting EM field (1). The charge separation results in a restoring force and subsequent coherent oscillation of the conduction band electrons, sharply enhancing scattering and absorption at specific resonance frequencies. The scattering spectra of nanoparticles are dictated by intrinsic parameters such as their size, shape, and composition, as well as extrinsic factors such as the local refractive index of the surrounding medium. These intrinsic parameters enable the design of gold nanoparticles with peak resonances that can be tuned to specific wavelengths across a relatively wide spectral region (600-2,200 nm) simply by modifying the synthesis protocol to realize the appropriate structural parameters (2,3). Because slight changes in the local refractive index of nanoparticles result in a peak wavelength shift, noble metal nanoparticles are also exquisitely sensitive reporters of their local, nanoscale environment.The unique properties of p...

Fast wide-field photothermal and quantitative phase cell imaging with optical lock-in detection

“…Following 20 minutes of incubation at 37 °C, the antibody-NP solution was removed, and cells were washed with media twice before imaging [13,14]. The same protocol was previously tested and validated by confirming anti-EGFR specific binding of nanoparticles to EGFR positive cancer cells with appropriate control experiments [3, 13–19]. Mie theory simulations indicate a substantially larger absorption cross section for 60 nm gold nanospheres as compared to lower diameter nanospheres, all of which have an absorption peak near 532 nm, the wavelength of the heating beam.…”

Optimization of immunolabeled plasmonic nanoparticles for cell surface receptor analysis

“…For example, silver nanospheres can scatter light from 400–500 nm while gold nanospheres predominantly scatter light from 500–600 nm. This range is further extended by gold nanorods, which can be adjusted with peak resonant wavelengths anywhere between 600–2200 nm [14]. …”

“…Simulations and experimental data have shown that there is a linear relationship between a GNR’s aspect ratio and its peak resonant wavelength [3, 24]. The longitudinal peak resonance can extend anywhere from 600 nm to 2200 nm [14]. This large range allows for the use of GNRs in many applications using both visible and NIR light.…”

“…Application of plasmonic nanoparticles can be somewhat limited due to their larger size, as compared to fluorescent dyes, providing a practical limit on the concentration delivered to cells and tissues. Upon antibody conjugation, immunolabeled plasmonic nanoparticles can be used to target specific molecules for sensing [9–14] and imaging [15–21] applications.…”

Physics of a rapid CD4 lymphocyte count with colloidal gold

“…Siiman et al noted that they were unsuccessful in creating side scatter clusters with nanoparticles smaller than 50 nm and speculated that other reports of side scatter cluster separation with 40‐nm gold particles may have been the result of accidental particle aggregation. More recently, Crow et al have reported the use of 30 nm rod‐shaped immunogold particles in a cellular assay (4). The method uses 20 min of vortex mixing.…”