title: Bioconjugated lanthanide luminescent helicates as multilabels for lab-on-a-chip detection of cancer biomarkers.
abstract: The lanthanide binuclear helicate [Eu(2)(L(C2(CO(2)H)))(3)] is coupled to avidin to yield a luminescent bioconjugate EuB1 (Q = 9.3%, tau((5)D(0)) = 2.17 ms). MALDI/TOF mass spectrometry confirms the covalent binding of the Eu chelate and UV-visible spectroscopy allows one to determine a luminophore/protein ratio equal to 3.2. Bio-affinity assays involving the recognition of a mucin-like protein expressed on human breast cancer MCF-7 cells by a biotinylated monoclonal antibody 5D10 to which EuB1 is attached via avidin-biotin coupling demonstrate that (i) avidin activity is little affected by the coupling reaction and (ii) detection limits obtained by time-resolved (TR) luminescence with EuB1 and a commercial Eu-avidin conjugate are one order of magnitude lower than those of an organic conjugate (FITC-streptavidin). In the second part of the paper, conditions for growing MCF-7 cells in 100-200 microm wide microchannels engraved in PDMS are established; we demonstrate that EuB1 can be applied as effectively on this lab-on-a-chip device for the detection of tumour-associated antigens as on MCF-7 cells grown in normal culture vials. In order to exploit the versatility of the ligand used for self-assembling [Ln(2)(L(C2(CO(2)H)))(3)] helicates, which sensitizes the luminescence of both Eu(III) and Tb(III) ions, a dual on-chip assay is proposed in which estrogen receptors (ERs) and human epidermal growth factor receptors (Her2/neu) can be simultaneously detected on human breast cancer tissue sections. The Ln helicates are coupled to two secondary antibodies: ERs are visualized by red-emitting EuB4 using goat anti-mouse IgG and Her2/neu receptors by green-emitting TbB5 using goat anti-rabbit IgG. The fact that the assay is more than 6 times faster and requires 5 times less reactants than conventional immunohistochemical assays provides essential advantages over conventional immunohistochemistry for future clinical biomarker detection.
authors.affiliation: Laboratory of Lanthanide Supramolecular Chemistry, Ecole Polytechnique Fédérale de Lausanne, BCH 1402, CH-1015 Lausanne, Switzerland.
authors.forename: Vanesa
authors.initials: V
authors.lastname: Fernández-Moreira
authors.forename: Bo
authors.initials: B
authors.lastname: Song
authors.forename: Venkataragavalu
authors.initials: V
authors.lastname: Sivagnanam
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Chauvin
authors.forename: Caroline D B
authors.initials: CD
authors.lastname: Vandevyver
authors.forename: Martin
authors.initials: M
authors.lastname: Gijs
authors.forename: Ilkka
authors.initials: I
authors.lastname: Hemmilä
authors.forename: Hans-Anton
authors.initials: HA
authors.lastname: Lehr
authors.forename: Jean-Claude G
authors.initials: JC
authors.lastname: Bünzli
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D001360:Avidin / Q000138:chemical synthesis / Q000737:chemistry; D014408:Biomarkers, Tumor / Q000032:analysis*; D045744:Cell Line, Tumor; D006367:HeLa Cells; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D007211:Indoles / Q000737:chemistry; D056656:Lab-On-A-Chip Devices; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D057230:Limit of Detection; D049408:Luminescent Agents / Q000737:chemistry*; D046210:Microfluidic Analytical Techniques; D010848:Picolinic Acids / Q000737:chemistry; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D001360:Avidin; D014408:Biomarkers, Tumor; D007211:Indoles; D028581:Lanthanoid Series Elements; D049408:Luminescent Agents; D010848:Picolinic Acids; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; C508053:ERBB2 protein, human