A 3D microfluidic paper-based electrochemical immunodevice (μ-PEID) for simultaneous sensitive detection of two tumor biomarkers was fabricated. A nanoporous Pt particle modified paper working electrode (NPPt-PWE) was used as the matrix and metal ions loaded on the l-cysteine capped flower-like Au nanoparticles (Au-Cys) were used as a signal amplification label. The NPPt-PWE was constructed through the growth of a NPPt layer on the back of the paper working electrode to improve electron conduction. Besides, the prepared flower-like Au-Cys could significantly enhance the amount of loaded metal ions and played an important role in connecting the signal antibodies. The metal ion labels could be detected directly through differential pulse voltammetry without metal pre-concentration, and the distinct voltammetric peaks had a close relationship with each sandwich-type immunoreaction. Therefore, a novel 3D μ-PEID for simultaneously detecting carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) with wider linear ranges was developed. The linear range was from 0.004 to 200 ng mL and the limits of detection for AFP and CEA were 1.0 pg mL and 1.3 pg mL (S/N = 3), respectively. The good stability, reproducibility, and accuracy of the μ-PEID indicate that it has great potential application in clinical diagnostics.
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