• 2019-07
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  • 2020-08
  • 2021-03
  • br Electrochemical characterization of cytosensor br In


    3.4. Electrochemical characterization of cytosensor
    In the Nyquist plot, the charge-transfer resistance (Rct) of the
    Fig. 1. (A) TEM image of PCN-224 (The inset shows the image of PCN-224 in aqueous solu-tions). (B) TEM image of PCN-224-Pt (The inset shows the image of PCN-224-Pt in aqueous solutions). (C) SEM image of PCN-224. (D) The dynamic light scattering (DLS) size distribution of PCN-224. (For interpretation of the refer-ences to colour in the text, the reader is re-ferred to the web version of this article.)
    Fig. 2. Agarose gel electrophoresis analysis of TDNs. Lane 1: DNA ladder
    electrode interface is equal to the semicircle diameter of the EIS. As depicted by the EIS in Fig. S6A, when TDNs, MCH, Lovastatin and nanoprobes were assembled on the bare GE, the semicircle diameter of EIS in-creased gradually, indicating that the Rct increased gradually, thus the architecture of the aptamer-cell-nanoprobe sandwich-like stepwise was accomplished successfully.
    Furthermore, DPV plot was demonstrated the signal amplification process of the nanoprobe (Fig. S6B). After capturing cancer cells, the GE exhibited a quite weak DPV peak current without nanoprobes for signal amplification (curve a). However, the DPV response increased ob-viously when the nanoprobes without hemin captured cells (curve b), attributing to the efficient electrocatalysis of HRP and PCN-224-Pt greatly accelerated the electrons transfer. Remarkably, the DPV signal further enhanced after using the nanoprobes with hemin to form G-quadruplex, which was ascribed to the catalysis of GQH DNAzyme (curve c). The results declared that we can use the functional hybrid nanoprobe including HRP, PCN-224-Pt and GQH DNAzyme to amply the signal of cytosensor.
    In addition, characterization of DPV response was applied the signal amplification strategy in different PBS solution (Fig. S6C). When the electrode was put into the PBS buffer, there is no electrochemical signal peak (curve a). A weak DPV reduction peak appeared when the elec-trode was immersed in the PBS buffer with only 3 mM HQ (curve b), ascribing to the electrochemical reduction of BQ. When the electrode was placed in the PBS buffer with 1.5 mM H2O2 and 3 mM HQ, a large DPV signal was observed (curve c), which was benefited from benzo-quinone (BQ) producing through the oxidation of HQ with H2O2. On the basis of the results above, the nanoprobe exhibited excellent peroxidase activity by availably catalyzing the oxidation of HQ with H2O2.
    3.5. Optimization of the experimental conditions
    Considering the multiple binding sites of the MCF-7 cell for the aptamers, both MUC1 and AS1411 could be used as the capture probes.
    Fig. 3. (A) Different proportions of the aptamers MUC1 and AS1411, including the ratio of (a) 0:1, (b) 1:0, (c) 1:1, (d) 1:2, (e) 2:1. (B) The current response of the cytosensor with different cells incubation times. (C) The current response of the cytosensor with different nanoprobes incubation times. The concentration of cells are all 1 × 104 cells/mL. Error bars are relative standard deviation of three independent experiments.
    To obtain higher sensitivity, we used different proportions of the ap-tamers MUC1 and AS1411 (the ratio of 0:1, 1:0, 1:1, 1:2, 2:1) (Fig. 3A). Using the single aptamer confined the surface binding sites of MCF-7 cell, the detection probes reduced the response. Thus, the sensors based on single aptamer MUC1 or AS1411 showed relatively low responses (the ratio of 0:1, 1:0). We obtained the best response while using the MUC1 and AS1411 aptamer (the ratio of 1:1). Based on these findings, we selected a MUC1 and AS1411 aptamer in 1:1 ratio to prepare the cytosensor for further experiments.
    We investigated the incubation time of cells on the electrode, and the GE was incubated in cells solution for different times (30, 40, 50, 60, and 70 min). The maximum signal of the cytosensor was reached in 60 min (Fig. 3B). It may keep the activity of cells and binding ability of cells to aptamer. Hence, 60 min was the best incubation time of cells in subsequent experiments. Lovastatin We also studied the incubation time (40, 50, 60, 70 and 80 min) of nanoprobes on the analytical performance about the DPV responses. As shown in Fig. 3C, the signal enhanced with in-creasing nanoprobes incubation time from 40 to 70 min and reached a turning point at 70 min. Thus, the optimal nanoprobes incubation time was 70 min in this cytosensor.