Sensitive Time-Gated Immunoluminescence Detection of Prostate Cancer Cells Using a TEGylated Europium Ligand

Abstract

We describe the application of a synthetically developed tetradentate β-diketonate-europium chelate with high quantum yield (39%), for sensitive immunodetection of prostate cancer cells (DU145). MIL38 antibody, a mouse monoclonal antibody against Glypican 1, conjugated directly to the chelate via lysine residues, resulted in soluble (hydrophilic) and stable immunoconjugates. Indirect labeling of the antibody by a europium chelated secondary polyclonal antibody and a streptavidin/biotin pair was also performed. All of these bright luminescent conjugates were used to stain DU145 cells, a prostate cancer cell line, using time gated luminescence microscopy for imaging, and their performances were compared to conventional FITC labeling. For all prepared conjugates, the europium chelate in conjunction with a gated autosynchronous luminescence detector (GALD) completely suppressed the cellular autofluorescence background to allow capture of vivid, high contrast images of immune-stained cancer cells.

Time-gated fluorescence imaging: Advances in technology and biological applications

“…Depending on the luminescence lifetime of the target probe, one may choose proper instrumentation for TG imaging. For quite long luminescence lifetime (e.g., lanthanide-based probes) up to millisecond time scale, 32,71,72,74,76,78,81 simple and low-cost instrumentation (mainly optical-chopper-based methods) is well satisfactory. By contrast, for quite short luminescence lifetime down to nanosecond time scale, 28,29,31,33,34,47,66 complex and expensive instrumentation (mainly electronic-unit-based methods) must be employed.…”

“…Recovery of the concealed micro objects such as large molecules, 11,28 cells, 11,23,29,34,36,39,41,47,49,66,70,72,[74][75][76][77][78][79][80][81][82][83][84] and microorganisms 24,30,32,71,85 from annoying interference (e.g., background AF) in°uorescence imaging with the help of TG technique has attracted increasing attention for years. Marriott et al presented a simple TG microscope and utilized it to visualize the delayed luminescence intensities and lifetime of acridine dyes on polytene chromosomes.…”

“…In 1990, imaging of living cells has been extensively demonstrated. To name a few, Madin Darby Canine kidney (MDCKII) cells, 79 human breast cancer cells, 66 human prostate cancer cells and bladder cancer cells, 78 HeLa cells (Figs. 8(a) and 8(b)), 34,41,81,84 HepG2 cells, 72,75,76,83,84 SKOV3 cancer cells, 50 AtT20 cells ( Fig.…”

Reduced background autofluorescence for cell imaging using nanodiamonds and lanthanide chelates

“…Luminescent compounds with long excited lifetimes enable time-gated image acquisition to be performed, eliminating cellular autofluorescence and thereby increasing the sensitivity of detection. Lanthanide metals, in the form of europium-chelating tags, are suitable for these time-gated imaging applications 13 , 14 . The streptavidin/biotinylated ligand system can be used with time-gated microscopy via a biocompatible europium chelating tag (such as BHHTEGST) covalently attached to the amines of the lysine residues in streptavidin (SA-BHHTEGST-Eu, Fig.…”

“…The streptavidin/biotinylated ligand system can be used with time-gated microscopy via a biocompatible europium chelating tag (such as BHHTEGST) covalently attached to the amines of the lysine residues in streptavidin (SA-BHHTEGST-Eu, Fig. 1aii ) 13 , 14 . Although the toxicity of lanthanide-containing tags is not yet established, these tags are particularly helpful for in vitro and ex vivo imaging where the background autofluorescence level is high.…”