Mapping Receptor Density on Live Cells by Using Fluorescence Correlation Spectroscopy

Abstract

Study of the density, spatial distribution, and molecular interactions of receptors on the cell membrane provides the knowledge required to understand cellular behavior and biological functions, as well as to discover, design, and screen novel therapeutic agents. However, the mapping of receptor distribution and the monitoring of ligand–receptor interactions on live cells in a spatially and temporally ordered manner are challenging tasks. In this paper, we apply fluorescence correlation spectroscopy (FCS) to map receptor densities on live cell membranes by introducing fluorescently marked aptamer molecules, which specifically bind to certain cell-surface receptors. The femtoliter-sized (0.4 fL) observation volume created by FCS allows fluorescent-aptamer detection down to 2 molecules and appears to be an ideal and highly sensitive biophysical tool for studying molecular interactions on live cells. Fluorophore-labeled aptamers were chosen for receptor recognition because of their high binding affinity and specificity. Aptamer sgc8, generated for specific cell recognition by a process called cell systematic evolution of ligands by exponential enrichment, was determined by FCS to have a binding affinity in the picomolar range (dissociation constant Kd = 790 ± 150 pM) with its target membrane receptor, human protein tyrosine kinase-7 (PTK7), a potential cancer biomarker. We then constructed a cellular model and applied this aptamer–receptor interaction to estimate receptor densities and distributions on the cell surface. Specifically, different expression levels of PTK7 were studied by using human leukemia CCRF-CEM cells (1300±190 receptors μm−2) and HeLa cervical cancer cells (550±90 receptors μm−2). Competition studies with excess nonlabeled aptamers and proteinase treatment studies proved the validity of the density-estimation approach. With its intrinsic advantages of direct measurement, high sensitivity, fast analysis, and single-cell measurement, this FCS density-estimation approach holds potential for future applications in molecular-interaction studies and density estimations for subcellular structures and membrane receptors.

Cell-Specific Aptamer Characterization

“…For example, when various concentrations of fluorescein isothiocyanate (FITC)-labeled sgc8 aptamers are incubated with HeLa cells, the different diffusion times of bound versus free aptamers can be used to assess the percentage of bound aptamer, followed by yielding the absolute number of total aptamers inside the confocal volume. A picomolar range dissociation constant (K d = 790 ± 150 pM) of sgc8 aptamer has been measured for HeLa cells, which is in good agreement with the result determined by flow cytometry (K d = 810 pM) [33].…”

“…Aptamers, which target cellular membrane surface biomarkers, are considered to be better molecular probes for surface biomarkers discovery compared to antibody-based recognition. This is due to the higher binding affinity (low K d ), specificity, and increased tumor penetration associated with the small size of aptamers, as well as the potentially reduced immunogenicity [32,33,36]. The density investigation of cellular receptors provided by the binding events of aptamers to specific receptors can help us to gain insight into membrane receptor characteristics, expression levels, and spatial distribution on the molecular level, as well as receptor clustering and molecular changes in living biological specimens.…”

“…4.2b). Similarly, a higher aptamer binding density is obtained (1,300 ± 190 receptors/µm 2 ) on CCRF-CEM leukemia cells [33]. FCS measurement provides a simple, direct, and accurate estimation of aptamer binding density on the cellular membrane surface, which opens a door for the investigation of drug delivery and efficiency assessment in cancer therapeutics.…”

“…However, due to the shorter half-life of the receptorligand complex compared to the time needed to separate bound and unbound ligands, this method sometimes causes an underestimate of the receptor density. The other concern is the low expression level of some receptors and interferences from other highly expressed receptors, resulting in an undetectable specific binding [33]. Thus, the development of a highly sensitive and effective approach for direct measurement of membrane receptor density on the cellular surface without radioactive labeling or tedious separation procedures is highly desired.…”

SERS-fluorescence dual mode nanotags for cervical cancer detection using aptamers conjugated to gold-silver nanorods

“…In the present nanotags, Au-Ag NRs were applied as the SERS substrates, and 4-ATP molecules were absorbed onto the NRs to provide SERS signal. The sgc8c aptamers on the nanotags can specifically bind to the PTK-7, which is overexpressed on Hela cell surface with the density of 550±90 receptor μm −2 based on the previous fluorescence correlation spectroscopy (FCS) study [30]. The high density of PTK-7 induced the accumulation of the nanotags into close proximity on the cell surface.…”