Electrochemical Biosesor Used To Detect MicroRNA-126 In Patients With Bladder Cancer

Objective:MicroRNA (miRNA) has emerged as new biomarkers for cancers in physiological fluids such as saliva, body fluids, and urine, whose prominent characters exist in their stability, noninvasive process, and early diagnosis. Deregulation of microRNA-126 (miRNA-126) is associated with bladder cancer. Accordingly, finding an appropriate way to monitor miRNA-126 in urine to make a definite diagnosis for bladder cancer has attracted much attention worldwide.We described a strategy for the determination of miRNA-126 that was based on the use of a glassy carbon electrode modified with carboxy-terminated generation 3.5 poly (amidoamine) (PAMAM) dendrimer, gold and silver nanoclusters, and a chitosan-graphene composite. This biosensor displays good reproducibility, stability and selectivity.Methods:1. Graphene sheets used to accelerate the electron transfer were added onto the glassy carbon electrode (GCE). It was important that chitosan-graphene promoted the biopolymer PAMAM-gold and silver nanoclusters composite (PAMAM-Au-Ag) to settle, as well as made the biosensor more stable.2. The PAMAM-Au-Ag layer could supply more active surface areas to capture the special hairpin peptide nucleic acid (PNA), consequently anchored more miRNA-126 to amplify the electrochemical signal. Then, the other side of the opened hairpin PNA was hybridized with the signal DNA labeled with digoxin.3. The typical sandwich structure was fabricated. Rabbit anti-digoxin/HRP was employed to provide the detection signal by the reaction with H2O2. The analytical signal was characterized by differential pulse voltammetry (DPV).4. Optimize the experimental conditions:the concentration of hairpin PNA, signal DNA concentrations, the ratio of rabbit anti-digoxin/HRP and the concentration of H2O2.5. Under the optimal environment, the developed biosensor was incubated in different concentrations of the target miRNA-126 solution to prepare the standard curve. Reproducibility, selectivity and stability were also analyzed in detail.Results:1. The UV-vis spectrometry of PAMAM, PAMAM-Au, PAMAM-Ag, and PAMAM-Au-Ag was shown to verify the successful synthesis. The scanning electron microscopy (SEM) and the transmission electron microscope (TEM) were also confirmed the modified GCE. Cyclic voltammograms (CVs) and electrochemical impedance spectroscopy (EIS) were simultaneously recorded to characterize the modification processes.2. The DPVs in the presence of different target miRNA-126 concentrations under the optimized conditions in 5 mM [Fe(CN)6]3-/4- and 0.1 M KC1 solution (pH 7.4). The calibration curve displayed a good linear relationship between the peak current and logarithm figures of the target concentrations in the range from 1.0 fM to 10 nM (the relative standard deviation was 3%). The corresponding equation can be expressed as I (μA)=5.336 log [C/M]+99.102 (R2=0.985). The detection limit was 0.79 fM.3. The immunosensor exhibited good reproducibility, selectivity, regeneration ability and stability.Conclusions:In summary, this novel electrochemical approach employed to determine the different levels of miRNA-126 was composed of PAMAM-Au-Ag and chitosan-graphene, which exhibited a broad dynamic range from 1.0 fM to 10 nM and a low detection limit 0.79 fM. The amounts of capture probe PNA immobilized on the as-prepared biosensor were enlarged. The final current response was achieved by introducing the rabbit anti-digoxin/HRP catalytic system. In case of efficient selectivity, stability, and affinity, this method was a promising tool used in clinical test. In conclusion, the strategy provided a powerful and versatile tool in miRNA detection fields to diagnose cancers early.