RESUMO
BACKGROUND: It is estimated that over 50 % of human cancers are caused by mutations in the p53 gene. Early sensitive and accurate detection of the p53 gene is important for diagnosis of cancers in the early stage. However, conventional detection techniques often suffer from strict reaction conditions, or unsatisfied sensitivity, so we need to develop a new strategy for accurate detection of p53 gene with smart designability, multiple signal amplification in mild reaction conditions. RESULTS: In this study, CRISPR/Cas system is exploited in entropy-driven catalysis (EDC) and hybridization chain reaction (CHA) dual signal amplification sensing strategies. The products of both reactions can efficiently and separately activate CRISPR/Cas12a which greatly amplifies the fluorescent signal. The method has good linearity in p53 detection with the concentration ranged from 0.1 fM to 0.5 pM with ultra-low detection limit of 0.096 fM. It also showed good performance in serum, offering potentials for early disease detection. SIGNIFICANCE: The designed dual amplification dynamic DNA network system exhibits an ultra-sensitive fluorescence biosensing for p53 gene identification. The method is simple to operate and requires only one buffer for the experiment, and meanwhile shows smart designability which could be used for a wide range of markers. Thus, we believe the present work will provide a potential tool for the construction and development of sensitive fluorescent biosensors for diseases.
Assuntos
Sistemas CRISPR-Cas , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/genética , Sistemas CRISPR-Cas/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico , Técnicas Biossensoriais/métodos , DNA/química , DNA/genética , Limite de Detecção , Genes p53 , Hibridização de Ácido NucleicoRESUMO
In this paper, we designed a novel shared cathode bipolar electrode chip based on Ohm 's law and successfully constructed a dual-mode dual-signal biosensor platform (DD-cBPE). The device integrates ELISA, ECL, and ECL imaging to achieve highly sensitive detection and visual imaging of carcinoembryonic antigen (CEA). The unique circuit structure of the device not only realizes the dual signal detection of the target, but also breaks the traditional signal amplification concept. The total resistance of the system is reduced by series-parallel connection of BPE, and the total current in the circuit is increased. In addition, Au@NiCo2O4@MnO2 nanozyme activity probe was introduced into the common cathode to enhance the conductivity of the material. At the same time, due to the excellent peroxidase (POD) activity of NiCo2O4@MnO2, the decomposition of H2O2 was accelerated, so that more electrons flowed to the BPE anode, and finally the dual amplification of the ECL signal was realized. The device affects the current in the circuit by regulating the concentration of the co-reactant TPrA, thereby affecting the resistance of the system. Finally, different luminescent reagents emit light at the same potential and the luminous efficiency is similar. In addition, the chip does not need external resistance regulation, which improves the sensitivity of the immunosensor and meets the needs of timely detection. It provides a new idea for the deviceization of bipolar electrodes and has broad application prospects in biosensors, clinical detection, and environmental monitoring.
RESUMO
Gastric cancer (GC) is characterized by high tumor heterogeneity, increased surgical difficulty, and limited chemotherapy efficacy, and it is associated with a poor prognosis. The abnormal proliferation of cells involves abnormal activation of the PI3K/AKT/mTOR signaling pathway. Inhibition of this signaling pathway can inhibit tumor cell proliferation and induce cell apoptosis. This study evaluated the effect of PF-04979064, a dual inhibitor of PI3K and mTOR, on human GC cells. PF-04979064 significantly inhibited the proliferation of human gastric adenocarcinoma AGS cells and the undifferentiated GC cell line HGC-27, promoting cell apoptosis. Combination treatment with PF-04979064 and the GC first-line clinical drug 5-FU showed synergistic effects, and PF-04979064 markedly increased the sensitivity of GC cells to chemotherapy drugs. Western blot results showed that PF-04979064 significantly inhibited the PI3K/AKT/mTOR signaling pathway in GC cells, whereas RNA seq results demonstrated substantial alterations in gene expression profiles upon treatment with PF-04979064. This study provides insight into the effects of PF-04979064, thereby establishing a solid foundation for its potential clinical application in the treatment of GC.