A mutiplex cytosensor based on a dual electrochemiluminescence (ECL) signal system was fabricated for in situ and simultaneous detection of the expression levels of multiple cell-surface receptors, mannose and epidermal growth factor receptor (EGFR), using luminol-capped gold nanoparticles (Au@luminol) and CdS quantum dots (CdS QDs) as potential-resolved ECL nanoprobes. Two spatially resolved areas on indium tin oxide (ITO) electrodes were modified with polyaniline (PANI) by electropolymerization, on which gold nanoparticles (AuNPs) were attached to strengthen conductivity and stability of the sensing interface. Human mucin1 protein (MUC1) aptamer was immobilized onto AuNPs for capturing MUC1-positive MCF-7 cells. Au@luminol and CdS QDs as ECL nanoprobes were covalently linked with concanavalin A (ConA) and epidermal growth factor (EGF) to label MCF-7 cells on the two areas of the cytosensor separately. Compared to conventional multiplex biosensor, we demonstrated a novel analysis platform for the simultaneous detection of multiple cell-surface receptors; it could provide two sensitive and potential-resolved ECL signals during one potential scanning and avoid cross-reactivity between the two nanoprobes. The quantification of MCF-7 cells on the two spatially resolved areas could be achieved over the linear range from 10 to 1.0 × 10 cells mL with a detection limit of 20 cells mL. This multiplex cytosensor was further applied for simultaneous quantitative evaluation of the expression levels of mannose and EGFR on MCF-7 cells, revealed that the average numbers of mannose and EGFR per captured MCF-7 cell were 1.2 × 10 and 0.86 × 10 with the relative standard deviation of 5.3% and 4.2%, respectively. The multiplex cytosensor was capable of evaluating multiple cell-surface receptors, which would be beneficial to developing a better diagnostic tool for diseases.

Cytochrome c (cyt c) and caspase-9 were critical biomarkers in mitochondria-mediated apoptosis. A novel electrochemical immunosensor was developed for in situ analysis of cyt c and caspase-9 in the cytosol. Gold nanoparticle-polydopamine (AuNP/PDA) composites were used to fabricate the interface of the sensor. The anti-cyt c or anti-caspase-9 functionalized-immunosensor provided a biomimetic interface for immunosensing of cyt c or caspase-9 in Hela cells during apoptosis. The changes in the expression level of cyt c and caspase-9 in the cytosol upon curcumin-induced apoptosis were detected by using the proposed method, and also the influence of different concentrations and incubation times of curcumin-induced Hela cells was investigated. This method achieved a linear range (0.1-100 μM) for standard cyt c and caspase-9, with a detection limit of 0.03 ± 0.01 μM for standard cyt c and 0.08 ± 0.02 μM for standard caspase-9. Moreover, this method was used to detect cells which could detect as low as 100 cells which expressed cyt c and caspase-9, and also the results are in good agreement with standard flow cytometry analysis. The developed electrochemical immunosensor offered a simple and rapid approach for sensitive evaluation of apoptosis markers with considerable specificity and reproducibility, and also the developed strategy could be of great importance in clinical diagnosis and therapeutic research.

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