Determination of different forms of human interferon‐γ in single natural killer cells by capillary electrophoresis with on‐capillary immunoreaction and laser‐induced fluorescence detection

Abstract

A novel method for determining different forms of human interferon-gamma (IFN-gamma) in single natural killer cells was developed by capillary electrophoresis (CE) with on-capillary immunoreaction and laser-induced fluorescence (LIF) detection. Cells were perforated with digitonin and one single cell was electrokinatically introduced into the front end of a separation capillary. The monoclonal antibody labeled with fluorescein isothiocyanate of IFN-gamma was hydrodynamically injected into the front end of the capillary around the cell introduced. After the cell was lysed by ultrasonication, the front end of the capillary was used as a microreactor to allow different forms of IFN-gamma to process the immunoreaction with their labeled antibody. Finally, the complexes of different forms of IFN-gamma with their labeled antibody were separated and detected by CE with LIF detection with a limit of detection of zeptomoles (10(-21) mol).

Current techniques for single-cell lysis

“…Sonication has several limitations that keep it from being widely used for single-cell lysis including the requirement of more than 50 s for the lysis of cells such as lymphocytes, which can result in significant heating and thus denaturing of proteins, and excessive diffusion of the cell contents that can lead to difficulties in downstream detection ( Xue & Yeung 1996;Zhang & Jin 2004. However, when used after first treating the cell with a weak detergent such as digitonin, sonication can lyse cells within 3 s, allowing efficient separation of cell contents (Zhang & Jin 2004.…”

“…However, tools that are capable of harvesting large amounts of proteomic data from single cells remain rather limited, largely owing to the difficulty involved in dealing with the small volumes and quantities of analytes concerned. Despite the limitations, over the last decade or two, there has been significant progress in developing assays capable of determining levels of specific proteins (Zhang & Jin 2004Huang et al 2007) and interrogating enzyme activity in single cells (Meredith et al 2000;Li et al 2004;Ocvirk et al 2004). One of the most limiting aspects for these studies to date has been the controlled disruption of selected cells.…”

Microfluidic Sample Preparation for Single Cell Analysis

“…184 Pretreatment with a non-ionic detergent shortened the required time to only 3 seconds. 184 Additionally, heating can be prevented by utilizing short discontinuous bursts instead of continuous sonication and by sonicating in an ice water bath. A freeze-thaw cycle can also be used to lyse cells.…”

Recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices

“…One single cell and the monoclonal antibody labeled with fluorescein isothiocyanate of IFN-␥ were successively (in turn) electrokinetically introduced into the front end of a separation capillary that acted as a microreactor to allow different forms of IFN-␥ from the lysate of the cell to process the immunoreaction with their labeled antibody. The complexes of different forms of IFN-␥ with their labeled antibody were separated and detected by CE with LIF detection with a limit of detection of zeptomoles [21]. To overcome the diffusion of the analytes along the longitudinal axis of the capillary during the on-capillary immunoreaction with antibody in the capillary microreactor and improve the separation efficiency, an improved labeling technique-intracellular immunoreaction was later developed in their group by introducing FITC labeled anti-IFN-␥ monoclonal antibody into NK cells by electroporation.…”