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.

Synthesis and cell localization of self-assembled dinuclear lanthanide bioprobes

“…No decomplexation is observed after one week. We note that the [Eu 2 (L C2COOH ) 3 ] 3− helicate has been previously coupled with avidin, yielding a luminescent bioconjugate (Q = 9.3%, τ ( 5 D 0 ) = 2.17 ms), which was successfully used as a detection probe for immunocyto-and immunohistochemical analyses [23].…”

“…Finally, the cellular uptake of five Eu III helicates is monitored by time-resolved luminescence microscopy and their localization in HeLa cells established by co-staining experiments. groups to yield H 4 L C2COOH allowed us to bioconjugate it to SA and monoclonal antibodies in order to develop very successful detection of cancerous cells in human breast tissue sections, owing to dual microfluidics assays of the receptors that they express [23].…”

Rare earths: jewels for functional materials of the future

“…The helicate luminescence is detected in time-resolved mode and the analysis time is at least 5 times shorter than in the classical protocol, along with a substantial reduction in the reactant volumes used. 49 It is anticipated that these bioprobes have good future perspectives since they can be excited in the NIR by multiphoton processes 50 and introduced into silica nanoparticles. 51 Currently, main focus in the development of lanthanidebased bioprobes is the engineering of dual imaging probes, combining the power of magnetic resonance and optical imaging techniques, 52 as well as the design of nanoparticles for temperature sensing in the physiological range, allowing temperature mapping of cells and tissues.…”

Lighting up cells with lanthanide self-assembled helicates

“…The better selectivity of the Eu III bioconjugates is confirmed by statistical analysis of luminescence microscopy images [69]. When MCF-7 cells were grown in the microchannel, the detection limit (DL) is larger, but the lanthanide probes remain more sensitive with DL ¼ 0.6-0.7 versus 1.4 mg ml…”