Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Animales , Conducta Animal , Técnicas de Cultivo/instrumentación , Técnicas de Cultivo/métodos , Diseño de Equipo , Humanos , Inmunohistoquímica/instrumentación , Inmunohistoquímica/métodos , Microscopía/instrumentación , Microscopía/métodos , Neurobiología/instrumentación , Neurobiología/métodosRESUMEN
Separation and subsequent culturing of MCF-7 breast cancer cells on self-assembled protein-coated magnetic beads in a microfluidic chip is demonstrated. The beads were patterned in situ inside a sealed microfluidic channel using magnetic-field-assisted electrostatic self-assembly. Hereafter, they were grafted by exposure to a solution of 5D10 monoclonal antibodies (mAb) and fibronectin (FN), with the first being used for immunospecific cell capture and the latter being used for cell adhesion and growth. A solution of target MCF-7 cells mixed with Jurkat cells was brought inside the microchannel, leading to specific MCF-7 cell capture; the latter were then cultured and evidenced by cell immuno-luminescence.
Asunto(s)
Neoplasias de la Mama/patología , Técnicas de Cultivo de Célula/instrumentación , Separación Celular/instrumentación , Magnetismo , Técnicas Analíticas Microfluídicas , Microesferas , Biomarcadores/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Adhesión Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Células Jurkat , Mediciones LuminiscentesRESUMEN
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.
Asunto(s)
Biomarcadores de Tumor/análisis , Inmunoensayo/métodos , Elementos de la Serie de los Lantanoides/química , Sustancias Luminiscentes/química , Técnicas Analíticas Microfluídicas , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Avidina/síntesis química , Avidina/química , Línea Celular Tumoral , Células HeLa , Humanos , Inmunoensayo/instrumentación , Indoles/química , Dispositivos Laboratorio en un Chip , Límite de Detección , Ácidos Picolínicos/química , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismoRESUMEN
PDMS-based microfluidic devices combined with lanthanide-based immunocomplexes have been successfully tested for the multiplex detection of biomarkers on cancerous tissues, revealing an enhanced sensitivity compared to classical organic dyes.
Asunto(s)
Biomarcadores de Tumor/análisis , Dispositivos Laboratorio en un Chip , Elementos de la Serie de los Lantanoides/química , Mediciones Luminiscentes , Neoplasias/patología , Biomarcadores de Tumor/inmunología , Línea Celular Tumoral , Dimetilpolisiloxanos/química , Humanos , Inmunoensayo , Factores de TiempoRESUMEN
We propose an original concept for a sandwich immunoassay that is completely performed on-chip using streptavidin-coated beads as substrate. The latter are electrostatically self-assembled on aminosilane micropatterns at the bottom of a microfluidic channel. We use mouse IgG diluted in phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA) solution as target antigen. The fluorescent sandwich immunocomplex is formed on the beads during the operation of the chip both in stop-flow and continuous-flow modes. Target mouse IgG antigen is detected down to a concentration of 15 ng/mL in stop-flow mode and 250 pg/mL in continuous-flow mode, using only 1300 nL of sample volume. We also demonstrate the possibility of simultaneous detection of two different antigens in a PBS-BSA solution using a dual microfluidic channel structure.