Advances in immunoanalysis of single cells with capillary electrophoresis

Abstract

After completion of a large number of genome-sequencing projects, we are in the era of the ‘-omics’, such as genomics, proteomics, metabolomics, and glycomics. These ‘-omics’ demand high-throughput of measurement techniques for average evaluation of hundreds to thousands of cells or targeting analysis of the biomarkers amongst such complex samples. But single-cell analysis can still tell us individual differences, give us the details of the processes occurring inside single cells, and reveal the existence of subpopulations of cells whose unique activities and characteristics have hitherto been obscured by population averaging. Particular benefits of capillary electrophoresis (CE), including small sample size, high separation speed and efficiency, and compatibility with the cellular environment, make it a suitable tool for single-cell analysis. There are thousands of compounds in one cell and it is still quite challenging to target what we are interested in on the single-cell scale with CE. One solution is immunoassay with CE, which combines the selectivity of immunoassay and the high separation efficiency of CE and makes targeting analysis possible on the single-cell scale. Immunoassay has become a popular technique because of its ability to selectively target a compound at trace levels in the presence of potential interferences at much higher concentrations, and its selectivity is based on the binding affinity between the antibody and the antigen. Common immunoassay formats are time consuming and labor intensive. Several technologies based on immunoassay, such as high-performance immunoaffinity chromatography (HPIC) [1], the capillary enzyme-linked immunoassay [2], and CE/immunoassay [3], were developed to overcome the drawbacks of common immunoassay formats. Moreover, the technique of CE with immunoassay may be amenable to automation and allow complete detection within several minutes. After immunoreaction of antibody and antigen, a CE separation of the mixture should reveal two types of zone: one corresponding to the complex and the other corresponding to free antigen or free antibody. If the complex remains stable on the separation time scale, then it should be possible to qualify and quantify the antibody/ antigen in the mixture based on the complex formed and/or the decrease of free antigen/antibody. The adaption of CE/immunoassay for single-cell applications presents several challenges, because the amount of material in a single cell is in the range of fmol to zmol and a CE analysis is usually applied after immunoreaction is finished, which often takes several minutes. Dilution of the constituents in a single cell should be minimized as much as possible in order to allow efficient target detection after the immunoreaction. The injection end of the capillary and the cell itself provide possibilities as the immunoreaction environment. A capillary with a diameter of tens of micrometers limits the diffusion/dilution of the complex and antigen during immunoreaction. The cell itself is another choice because the cell membrane is an impermeable barrier and keeps essential metabolites and macromolecules inside the cell. Single-cell assay requires increasingly sensitive and selective detection technology. The detection methods often utilized in single-cell measurements are either laserinduced fluorescence (LIF) detection or electrochemical detection (ED). Since many antibodies and antigens, labeled with fluorescence reagents such as fluorescein isothiocyanate (FITC), are commercially available [4, 5], LIF detection method is commonly employed for single-cell measurement with CE/immunoassay. H. Zhang (*) . S. Tian State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, People’s Republic of China e-mail: zhanghua@sdu.edu.cn