Quantification and Imaging of Antigens on Cell Surface with Lipid-Encapsulated Fluorescent Nanodiamonds

Abstract

Quantifying the density and locating the position of antigens on cell surface has been a challenge in molecular biology research. The challenge lies in the need for a chemically and photophysically stable fluorophore to achieve the required sensitivity and accuracy. Here, we present a method suitable for the purpose by using lipid-encapsulated fluorescent nanodiamonds (FNDs) of 35 nm in diameter as biolabels. The encapsulation of FNDs in biotinylated phospholipids not only facilitates good dispersion of the particles in biological buffers, but also endows them with high specific targeting ability. We demonstrated a viable application of the technique for biotin-mediated immunostaining of antigens on fixed human cells, identifying their positions by two-color confocal fluorescence imaging, and determining their densities by magnetically modulated fluorescence detection. A binding capacity of 6 ± 1 × 104 antigens/cell was measured specifically for CD44 on HeLa cell surface. The result agreed well with the assay of R-phycoerythrin-conjugated antibodies by flow cytometry, supporting the reliability of this new nanoparticle-based method.

Nanodiamonds in biomedical research: Therapeutic applications and beyond

“…Heish et al (2019) demonstrated a system for determining the position and density of antigens on human cells using modulated FND detection. The results aligned with an assay of R-phycoerythrin-conjugated antibodies by flow cytometry, supporting the reliability of this approach ( 87 ). Similarly, FNDs have been shown viable for rapid detection of allergens in foods using a ND-based voltammetric immunosensing platform ( 91 ).…”

“…Rapid detection of viral exposure and infection represents a promising application for FNDs ( 87 , 88 ).…”

Multiplexed microfluidic chip for cell co-culture

“…The problem was simplified by considering the antibody binding region as the flat, bottom surface of the channel (neglecting the height of cells, and diffusion through the cytoplasm) (28)(29)(30)(31)(32) . Approximations were made for the density of antigens on the surface based on antibody binding capacity reported for cell surface antigens in the context of flow cytometry (33)(34)(35). We chose a value of 1×10 5 sites/cell.…”

Multiplexed microfluidic chip for cell co-culture

“…19–23 Approximations were made for the density of antigens on the surface based on antibody binding capacity reported for cell surface antigens in the context of flow cytometry. 24–26 We chose a value of 1 × 10 5 sites per cell. Antibody association and dissociation rates ( k on and k off ) were approximated as 1 × 10 5 s −1 m −1 and 1 × 10 –5 s −1 respectively.…”