Integrated bio-affinity nano-platform into a microfluidic immunosensor based on monoclonal bispecific trifunctional antibodies for the electrochemical determination of epithelial cancer biomarker.
BACKGROUND
The epithelial cell adhesion molecule (EpCAM) is a biomarker that is highly overexpressed on the surface of epithelial carcinoma cells. In this study, silver nanoparticles covered with polyvinyl alcohol (AgNPs-PVA) were synthesized, characterized and used in a microfluidic immunosensor based on the use of anti-EpCAM recombinant antibodies as a trapping agent.


METHODS
The concentration of trapped EpCAM is then electrochemically quantified by HRP-conjugated anti-EpCAM-antibody. HRP reacted with its enzymatic substrate in a redox process which resulted in the appearance of a current whose magnitude (at a working voltage as low as -0.10V) is directly proportional to the concentration of EpCAM.


RESULTS
Under optimized conditions, the detection limits for the microfluidic immunosensor and a commercial ELISA were 0.8 and 13.9pg/L, respectively. The within-assay and between-assay coefficients of variation are below 6.5% for the proposed method. The immunosensor was validated by analyzing patient samples, and a good correlation with a commercial ELISA was obtained.


CONCLUSIONS
The good analytical performance is attributed to the efficient immobilization of the anti-EpCAM recombinant antibodies on the AgNPs-PVA, and its high specificity for EpCAM. This microfluidic immunosensor is intended for use in diagnosis and prognosis of epithelial cancer, to monitor the disease, and to assess therapeutic efficacy.
INQUISAL, Departamento de Química, Universidad Nacional de San Luis, CONICET, Chacabuco 917, D5700BWS San Luis, Argentina.
Karina
K
Bravo
GENYO, Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, Avenida de Ilustración, 114 18016 Granada, Spain.
Francisco G
FG
Ortega
INQUISAL, Departamento de Química, Universidad Nacional de San Luis, CONICET, Chacabuco 917, D5700BWS San Luis, Argentina.
Germán A
GA
Messina
INQUISAL, Departamento de Química, Universidad Nacional de San Luis, CONICET, Chacabuco 917, D5700BWS San Luis, Argentina.
María I
MI
Sanz
INQUISAL, Departamento de Química, Universidad Nacional de San Luis, CONICET, Chacabuco 917, D5700BWS San Luis, Argentina. Electronic address: mbaldo@unsl.edu.ar.
Martín A
MA
Fernández-Baldo
INQUISAL, Departamento de Química, Universidad Nacional de San Luis, CONICET, Chacabuco 917, D5700BWS San Luis, Argentina.
Julio
J
Raba
D018033:Antibodies, Bispecific / Q000276:immunology*; D014408:Biomarkers, Tumor / Q000097:blood; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D003110:Colonic Neoplasms / Q000097:blood*; D004563:Electrochemistry; D000071858:Epithelial Cell Adhesion Molecule / Q000097:blood* / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D056656:Lab-On-A-Chip Devices; D057230:Limit of Detection; D053768:Metal Nanoparticles / Q000737:chemistry; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D012834:Silver / Q000737:chemistry
D018033:Antibodies, Bispecific; D014408:Biomarkers, Tumor; D000071858:Epithelial Cell Adhesion Molecule; D012834:Silver
Cancer diagnosis; Epithelial cell adhesion molecule; Microfluidic immunosensor; Nano-platform; Recombinant antibody; Tumor marker