An endonuclease-linked multiplex immunoassay for tumor markers detection based on microfluidic chip electrophoresis for DNA analysis

It is crucial to detect multiple tumor markers (TMs) for cancer diagnosis. In the study, an endonuclease-linked multiplex immunoassay for TMs detection, with alpha-fetoprotein (AFP), carcino-embryonic antigen (CEA), carbohydrate antigen 199 (CA199) as models, was developed using microfluidic chip electrophoresis (MCE) for DNA analysis as platform. Firstly, three endonuclease (EDOs) labels were prepared by labeling secondary antibodies of TMs with EDOs (BamHI, PstI, EcoRI), respectively. Then, the primary antibodies of TMs were co-immobilized on the bottoms of 96-pore plate. Finally, the TMs and labels were simultaneously incubated in the pore to form various sandwich immunocomplexes. The EDOs on them can cut their corresponding DNA substrate strands into half fragments which can be separated and detected by MCE. The signal ratio (Ih/Is) of half fragments (Ih) and substrate (Is) was proportional to logarithm of TMs concentrations from 1 pg mL−1 to 10 ng mL−1 (U mL−1 for CA199) with detection limits of 0.35, 0.3 pg mL−1 for AFP, CEA and 0.36 U mL−1 for CA199. The multiplex immunoassay was successfully employed in human serums. More importantly, based on the signal conversion mode, MCE for DNA analysis can be easily extended to detect many diagnosis markers and has applicable value in clinics.