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

Feng-Jen Hsieh,Yuen Hui,Chun-Hung Lin,Huan-Cheng Chang,Yen-Wei Chen

doi:10.3390/mi10050304

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.

Highlights

The surface of a cell is covered with various kinds of antigens [1]
Synthetic type Ib diamond powders (Micron + MDA) were obtained from Element Six; egg PC, cholesterol, 18:0 PEG2000 PE, and DSPE-PEG2000 biotin were from Avanti; Dulbecco’s modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS) were from Gibco; biotinylated anti-CD44 antibody was from Biolegend; NeutrAvidin and DyLight 488-conjugated NeutrAvidin were from Thermo Scientific; Atto542-biotin was from ATTO-TEC; paraformaldehyde was from Electron Microscopy Sciences; bovine serum albumin (BSA), tetrahydrofuran (THF), and all other chemicals were from Sigma-Aldrich and were used without further purification
These properties together make it possible to achieve high-quality, background-free imaging and localization of cellular components with nanoscale resolution if the nanoparticles are endowed with specific targeting abilities. This work demonstrated their feasibility by using bL-fluorescent nanodiamonds (FNDs) to determine the density of CD44 antigens on human cervical cancer line (HeLa) cell surface by magnetically modulated fluorescence (MMF) detection

Summary

Introduction

The surface of a cell is covered with various kinds of antigens [1]. These antigens are important molecular markers for the identification of different cell types and specific targets for the treatment of different diseases [2,3,4,5,6]. Human leukocyte antigens (or CD antigens), on the other hand, are proteins, and they play a central role in immune response [5] The significance of these membrane-bound molecules has stimulated the development of an immunoenzymatic technique known as cell-enzyme-linked immunosorbent assay (cell-ELISA) to achieve quantitative analysis of cell surface antigens [7,8,9]. Atomic force microscopy [10] and optical microscopy [11,12,13,14] serve the latter purpose well, but are unable to determine the antigen densities with sufficient accuracy Aiming to overcome these limitations, we have developed in this work a method that allows quantification of cell surface antigens, and their spatial localization with nanometric resolution. The key component of the technology is the lipid-encapsulated fluorescent nanodiamond (FND), which hosts a high-density ensemble of negatively charged nitrogen-vacancy (NV–) defects as fluorescent centers [15]

Methods

Results

Conclusion