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  • br Reproducibility and long term stability br

    2020-03-24


    3.7. Reproducibility and long-term stability
    The stability [email protected]–C/GCE and reproducibility of sensing assay are
    Fig. 6. (A–D) Confocal microscope images re-corded at an excitation wavelength of 488 nm and emission wavelength at 620 nm of control ABT-263 (Navitoclax) (Hela cells). A) Bright field image of [email protected] S-CMS incubated with Hela cells. B) Nucleus blue staining (DAPI) of Hela cells image. C) F-actine photo image of the Hela cells. D) Merged image for Hela cells upon incubation with [email protected] S-C. E) The amperometric response of H2O2 released from Hela cells within addition of 1 mM AA (green line) in 0.1 M PBS containing 6 × 105 cells/mL, the black line shows the addition of AA in 0.1 PBS without cells at ap-plied potential -0.35 V on the [email protected]/GCE (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
    key components for long-term, accurate and precise monitoring of targets. The stability of [email protected]–C/GCE was evaluated by measuring the prcise monitoring of H2O2 target within 7 times (≥7) under the op-timum sensing conditions. The RSD of chronoamperometric response assays (repated for ≥7 times) of 10 μM H2O2 on [email protected]–C/GCE reveled that the RSD was 2.7%, indicating the high stability of the designed electrode in H2O2 monitoring (Fig. S5A). Moreover, the electrode re-producibility can be approved by using freshily five [email protected]–C/GCE electrodes at a constant concentration of H2O2 (50 μM), in five optimum sensing assay. In this regard, each electrode was washed with 0.1 M PBS (pH 7). The concentration of H2O2 was measured through a chron-oamperometric method under optimum sensing conditions (Fig. S5B). The [email protected]–C/GCE electrode shows high-sensing response efficiency (i.e., 98%, with %RDS = 3.7), despite the 5th reuses/cycles of electrode usage in the H2O2 sensing assays. The designed [email protected]–C sensor is highly stable and reproducable. The electrode reusability and stability may show potential in its economic evaluation [53–55]. 
    3.8. Active signalling of ORS-released from cancer cells
    Due to the unique structural features of the designed [email protected]–C electrodes, the H2O2 sensing assay showed fast response time in a wide linear range of detection, high sensitivity and selectivity, low cyto-toxicity, high biocompatibility, and reasonable reproducibility and stability. For the control in vitro analysis, the [email protected]–C/GCE electrode design is examined to control an in vitro monitoring of H2O2 released from living cells such as HeLa cells. In this approach, human cervical carcinoma (HeLa cells) was selected for the detection of ORS released from stimulated cancer cells. The HeLa cell line represents a significant model for cervical cancer diagnosis and treatment [56].
    The biocompatibility of [email protected]–C was investigated for approaching the in vitro model for ORS secreted from cancer cells. Confocal mi-croscope Leica Microsystems LAS AF-TCS MP5 was used for cell ima-ging with [email protected]–C. Fig. 6 shows the confocal capturing images for HeLa cells after incubation with [email protected]–C for 2 h and staining with phalloidin and DAPI for F-actin and nucleus counterstaining, respectively.
    Confocal capturing of the bright photo (Fig. 6A), cytoskeleton (Fig. 6B), nucleolus (Fig. 6C), and merged photo between the nucleus and cell cytoskeleton (Fig. 6D) was performed. As illustrated from all captured photos, there is no any change in cell morphology and viability, leading to the high biocompatibility of [email protected]–C which can be advanced for in vivo studies.
    The continuous release of H2O2 in HeLa cells was detected by using [email protected]–C/GCE under H2O2 stimulation by AA [48]. Fig. 6E shows the amperometric response of [email protected]–C/GCE in the presence and absence of 6 × 105 HeLa cells in PBS at pH 7 and applied potential of –0.35 V and compared with that of Ag/AgCl. In the controlled experiments (black line, Fig. 6E), 1 mM AA was added into 0.1 M PBS (pH 7) at an applied potential of –0.35 V represents, where there is no observed response or current change of the amperometric signal. Under the same conditions, 1 × 106 HeLa cells in PBS at pH 7 were tested under stimulation of 1 mM AA (Fig. 6E green line]). A significant amperometric response was obtained, and the current reached its maximum value within a few seconds (5 s) upon the injection of 1 mM AA. These results may be at-tributed to the secretion of H2O2 from HeLa cells. The observed current was 3.5 μA for 1 × 106, matching with the calibration curve; each cell produces 16 ± 0.02 nM H2O2, which is near the detection limit of our designed electrode (25 nM). Our finding indicated that the [email protected]–C/ GCE can be employed for the ultrasensitive detection of H2O2 released from living cells under physiological conditions.