Bioconjugated lanthanide luminescent helicates as multilabels for lab-on-a-chip detection of cancer biomarkers

摘要

The lanthanide binuclear helicate [Eu2(LC2(CO2H))3] is coupled to avidin to yield a luminescentnbioconjugate EuB1 (Q ¼ 9.3%, s(5D0) ¼ 2.17 ms). MALDI/TOF mass spectrometry confirms thencovalent binding of the Eu chelate and UV-visible spectroscopy allows one to determinena luminophore/protein ratio equal to 3.2. Bio-affinity assays involving the recognition of a mucin-likenprotein expressed on human breast cancer MCF-7 cells by a biotinylated monoclonal antibody 5D10 tonwhich EuB1 is attached via avidin-biotin coupling demonstrate that (i) avidin activity is little affected bynthe coupling reaction and (ii) detection limits obtained by time-resolved (TR) luminescence with EuB1nand a commercial Eu-avidin conjugate are one order of magnitude lower than those of an organicnconjugate (FITC-streptavidin). In the second part of the paper, conditions for growing MCF-7 cells inn100–200 mm wide microchannels engraved in PDMS are established; we demonstrate that EuB1 can benapplied as effectively on this lab-on-a-chip device for the detection of tumour-associated antigens as onnMCF-7 cells grown in normal culture vials. In order to exploit the versatility of the ligand used for selfassemblingn[Ln2(LC2(CO2H))3] helicates, which sensitizes the luminescence of both EuIII and TbIII ions,na dual on-chip assay is proposed in which estrogen receptors (ERs) and human epidermal growth factornreceptors (Her2eu) can be simultaneously detected on human breast cancer tissue sections. The Lnnhelicates are coupled to two secondary antibodies: ERs are visualized by red-emitting EuB4 using goatnanti-mouse IgG and Her2eu receptors by green-emitting TbB5 using goat anti-rabbit IgG. The factnthat the assay is more than 6 times faster and requires 5 times less reactants than conventionalnimmunohistochemical assays provides essential advantages over conventional immunohistochemistrynfor future clinical biomarker detection.