Simultaneous detection of multiplex biomarkers related with hepatocellular carcinoma by coupling DNase I-assisted recycling amplification and microfluidic electrokinetic stacking chip with parallel multi-channels

Hepatocellular cancer (HCC) is the fourth most common death related cancer in worldwide. Given that simultaneous multiplex biomarker detection might significantly increase the sensitivity and specificity of HCC diagnosis. Herein, we developed a strategy by coupling 6-carboxyfluorescein labelled aptamer (FAM-Apt), reduced graphene oxide (rGO), deoxyribonuclease I (DNase I)-assisted recycling amplification and microfluidic electrokinetic stacking chip (FAM-Apt/rGO/DNase I-MESC) for detecting multiple HCC biomarkers including alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and microRNA-21 (miR-21). Thereinto, FAM-Apts were first adsorbed by rGO, resulting in fluorescence quenching, and then the FAM-Apts dissociated from rGO after binding the target. Subsequently, the FAM-Apts were degraded by DNase I and the targets were released and recognized by the FAM-Apts on the surface of rGO again. The cyclic dissociation and degradation of FAM-Apts produced FAMs and achieved primary signal amplification. Within 30 min, the released FAMs were accumulated and detected in MESC with three parallel micro-channels, resulting in secondary signal amplification. Based on FAMApt/rGO/DNase I-MESC, the limit of detection (LOD) for AFP, CEA and miR-21 were 37.0 pg/mL, 4.5 pg/mL and 1.3 fM, respectively. Remarkably, our method was successfully applied to detect AFP, CEA, and miR-21 in clinical serum samples. The levels of AFP and CEA achieved by our method had a good agreement with the enzyme-linked immunosorbent assay (ELISA) results. The sensitivities of AFP, CEA, miR-21 and the combination of the three targets for differentiating HCC patients from healthy individuals were 71.43%, 57.14%,85.71% and 100%, respectively, indicating the significance of the simultaneous detection of multiple indicators in clinic.

Automated summary

In this study, a strategy for detecting hepatocellular cancer biomarkers was developed. This strategy involved the coupling of a 6-carboxyfluorescein labelled aptamer, reduced graphene oxide, DNase I-assisted recycling amplification, and microfluidic electrokinetic stacking chip. The method demonstrated high sensitivity and specificity in detecting multiple biomarkers simultaneously, and was successfully applied to clinical serum samples.