Fig 1: Characterization of human-derived tumor spheroid. (A) Schematic diagram of spheroids for HT-29 cells and MSCs. (B) Immunofluorescence staining images of tumor spheroid and normal spheroid (X630). (C) Fluorescence expression area graph in tumor spheroids and normal spheroids (n = 3). (D) FACS intensity plot of tumor spheroid and normal spheroid for CEA. (E) FACS intensity plot of tumor spheroid and normal spheroid for Ki67. (F) Protein expression by ELISA assay from cultured medium (n = 5). (G) Real-time quantitative PCR graphs of tumor spheroids and normal spheroids (n = 3). Normal cell: MSC (mesenchymal stem cell), Tumor cell: HT-29 (Human colorectal adenocarcinoma cell). Scale bar: (B) 40 μm. All data represent mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. The symbol ∗ indicates comparisons with MSC.
Fig 2: Characterization of the electrochemical immunosensor at the microfluidic platform and in vitro detection of Ki67 and CEA. Schematic illustration: (A) fabrication process of anti-CEA and anti-Ki67 antibody functionalized immunosensor. (B) Printing process of resin master molds for microfluidic layer and chamber fabrication. (C) FE-SEM image of MWCNT, AuNP and AuNP@MWCNT. (D) Elemental mapping of MWCNT, AuNP and AuNP@MWCNT. (E) Cyclic voltammetry graph of immunosensor in each fabrication step (F) Peak current of each fabrication step. Normalized peak current of (G) anti-CEA antibody and (H) anti-Ki67 antibody functionalized immunosensor after incubating in various concentrations of antigen for 35 min (n = 3). Scale bar: (C) 200 nm, (D) 250 nm.
Fig 3: Schematic illustration: Detection process of antigens from cell culture medium with anti-Ki67 and anti-CEA antibody functionalized immunosensor.
Fig 4: Detection of CEA and Ki67 in spheroid medium. (A) Schematic illustrations of antigen detection from CT-26, MSC and HT-29 culture medium using the immunosensor. LSV measurements comparison of anti-CEA antibodies and anti-Ki67 antibodies functionalized immunosensor for (B) CT-26 vs MSC, (C) HT-29 vs MSC, and (D) Treat (Transfected) vs Control (Non-transfected) after incubating in various cell culture medium with a voltage sweep from −0.5 V to 0.5 V at a 50 mV/s scan rate (n = 3).
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