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  • br Scheme A Fabrication procedures of PCN Pt HRP dual

    2019-10-21


    Scheme 1. (A) Fabrication procedures of PCN-224-Pt/HRP/dual-aptamer/GQH nanoprobe. (B) Schematic illustration of the electrochemical dual-aptamer cyto-sensor fabrication process.
    staining.
    3. Results and discussion
    3.1. Principle of the electrochemical aptamer cytosensor
    The principle of the fabricated cytosensor is presented in Scheme 1. The synthetic PCN-224-Pt nanostructures as the nanocarriers were in-troduced for increasing the amount of biocatalysts. Due to the elec-trostatic attraction, Pt nanoparticles were decorated on the surface of the PCN-224 nanomaterials (Fig. S1). Subsequently, the thiolated cap-ture probes (containing the sequences of AS1411 or MUC1 aptamer (Fig. S2) and G-quadruplex) and the moderate HRP were decorated on the PCN-224-Pt by the thiolemetal chemical bond forming DNA-mod-ified nanoprobes. The catalytic HRP-mimicking GQH DNAzymes were assembled on the probes with the presence of hemin and potassium ion in PBS buffer. The electrochemical hybrid nanoprobes consist of the immobilization of GQH DNAzyme, HRP and dual-aptamer on the sur-face of PCN-224-Pt nanoparticles (Scheme 1A).
    Then the thiolated TDNs linked dual-aptamer AS1411 and MUC1 were attached to the GE surface via goldethiol bond to capture MCF-7 (-)-Bicuculline methiodide specifically. Afterward, the electrochemical cytosensor was further assembled with aptamer-cell-nanoprobe forming the sandwich struc-ture based on the special affinity. The signal amplification method of the cytosensor was catalyzed by PCN-224-Pt, HRP and GQH DNAzyme, which was carried on the electrochemical feature of production ben-zoquinone (BQ) from the oxidation of HQ with H2O2. The amount of electrochemical nanoprobes on the GE was directly related to signal of the electrochemical cytosensors which could directly reflect the amount of cells captured on the TDNs-modified GE by the aptamers (Scheme 1B).
    3.2. Characterization of nanoprobes
    As we can see the scanning electron microscopy (SEM) image in Fig. 1A, the dark purple products PCN-224 are isolated and spherical nanoscale particles. As shown in Fig. 1B, a monolayer of dark green 
    nanoparticles with a diameter of about 2 nm was covered on the iso-lated and spherical nanoparticles to form a core-satellite nanostructure. As seen by the transmission electron microscope (TEM) diagram of the PCN-224 (Fig. 1C), the products are all spherical and uniformly dis-persed structures. Besides, the result of Fig. 1D confirms that the size of PCN-224 is a narrow particle size distribution with 95 nm in diameter. In addition, the SEM elemental mappings (Fig. S3) indicated the Zr and Pt elements were distributed in the same particle, which further proved the Pt nanoparticles were decorated on PCN-224. Meanwhile, the en-ergy-dispersive spectroscopy (EDS) of Fig. S4A and B showed that the diagram of PCN-224-Pt appeared the obvious peaks of the Pt element. All of the pictures manifested the PCN-224-Pt had been synthesized. After successful synthesis of the PCN-224-Pt nanocomposites, their catalytic properties were then verified. H2O2 could be decomposed by catalase to generate oxygen, thus the tubes containing catalase and H2O2 appeared the gas bubbles. Similarly, the gas bubbles also were produced in the tubes containing H2O2 and PCN-224-Pt.There formed no gas bubbles in the control experiment (Fig. S5). It proved that it was PCN-224-Pt that had catalase-like activity. Taking together, all of the above results indicated that PCN-224-Pt had been successfully synthe-sized with catalase-like activity.
    3.3. Agarose gel electrophoresis analysis of TDNs
    TDNs linked dual aptamers were introduced to enhance detection selectivity and sensitivity. The result of agarose gel electrophoresis was employed for the characterization of the assembly of TDNs. The greater the molecular weight, the slower the migration. After separation, the gel was imaged using the fluorescence gel imaging system (Fig. 2). The assembled TDNs (lane 2 and 3) moved (-)-Bicuculline methiodide more slowly than other struc-tures formed by less than four single-stranded oligonucleotides (lane 4–6) because of the TDNs with more bases. The results demonstrated the successful self-assembly of TDNs in the work.