Highly Sensitive Protein Detection Based on Lanthanide Chelates with Antenna Ligands Providing a Linear Range of Five Orders of Magnitude

Abstract

Protein detection is an important task for pharmaceutical and clinical research today. Numerous protein staining techniques exist but are limited regarding their sensitivity and often narrow linear quantification ranges. To the best of our knowledge, this is the first description of a novel class of lanthanide chelatators, which absorb in the lower energy region at 360 nm. The new compound (6,9-dicarboxymethyl-3-{4-([1,10]-phenanthrol-2-ylethinylphenyl-carbamoyl)-methyl}-3,6,9-triaza-)-undeca-1,11-dicarboxylic acid) was coupled to different proteins and showed highly sensitive protein detection limits in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1.5 fmol of bovine serum albumin) as well as Dot Blot (100 amol of lysozyme). Furthermore, the novel compound shows an exceptionally broad linear quantification range over 5 orders of magnitude allowing applications that require the highest sensitivity alongside standard sensitivity. In addition, the new compound offers multiplexing capabilities.

Lanthanide-based time-resolved luminescence immunoassays

“…By comparison, the pendant chromophore strategy involves the grafting of one or more chromophores on to one of the aforementioned multidentate chelators, for example DTPA or DO3A. Two examples of ligands developed using this strategy are DTPA-cs124 ( ε  ≈ 8000 mol −1  L cm −1 (337 nm)) [16] and [EuL]H [17]. Although both strategies can lead to effective chelators, the chromophoric chelate approach is considered preferable because it places the antenna function in close proximity to the metal and thereby facilitates rapid energy transfer [18].…”

Stimuli-responsive electrospun fibers and their applications

“…86,87 To this end, a lot of effort is put in optimizing the sensitivity of protein detection, e.g. by developing novel staining techniques for Western blots 88 or by introducing completely new detection methods based on, as an example, gold nanoparticles. 89 These new techniques still suffer from important drawbacks in terms of limited sensitivity, poor linearity, or low ability for high-throughput analysis.…”

His-tag protein monitoring by a fast mix-and-measure immunoassay

“…Especially in case of non-automated, manual fraction collection in chromatography, the workload to identify the His-tag containing fractions via SDS-PAGE and Western blot is significant. In this assay, the protein sample is mixed with a small His-peptide that is labeled with the phosphorescent donor dye EuLH10 - a phenanthroline-based europium chelate - and then briefly incubated with the anti-His-tag antibody “8-4-4”, which was labeled with Black hole quencher 10 (BHQ-10) as an acceptor dye for EuLH. The target protein competes with the EuLH donor peptide for the paratope binding site of the antibody, resulting in a target-concentration-dependent phosphorescence signal (also referred to as time-resolved fluorescence, TRF).…”

“…The unique properties of the used lanthanide dye EuLH, i.e. that it can be excited in the near-Vis region at 360 nm, enables the use of simple and cheap glass optics instead of quartz glass for the measurement10. The use of a His 10 - instead of a His 6 - tag could potentially improve the detection limit of the assay described here due to a better sterical accessibility of the epitope.…”

“…EuLH10 and carboxyfluorescein as donor in the homogeneous assay were incorporated to the free N-terminus via a free carboxyl group using the same reaction conditions as for each amino acid coupling before. All peptides were cleaved from the synthesis resin with TFA and finally purified via RP-HPLC and characterized using MALDI-MS.…”