Reserch On Label Free Biosensor Technology And Construction Of Detection System For Water Samples

The detection for environmental pollutant has become the key indicator for environmental evaluation and protection. The development of the biosensor to detect environmental pollutant rapidly and effectively shows great scientific significance and promising application. In this study, 2 novel biosensors were constructed for the determination of heavy metal copper ions and kanamycin, and their detection process and mechanism were also investigated. The results were as follows:1)A simple, rapid and highly sensitive strategy was developed based on Fenton reaction and unmodified gold nanoparticles(AuNPs) for the detection of Cu2+ in water samples. The hydroxyl radical(·OH) generated by the Fenton reaction between the Cu2+ and sodium ascorbate oxidized the single stranded DNA(ssDNA) attached on the AuNPs surface into different sequence fragments. The cleavage of ssDNA induced the aggregation of AuNPs in a certain salt solution, therefore, resulting in the changes on the absorbance of solution. Experimental results showed that under the conditions of pH 7.9, 11 mg/L ssDNA,8 mm/L SA and 70 mmol/L NaCl,the absorbance ratio values at the wavelengths of 700 and 525 nm(A700/A525) were linearly correlated with the Cu2+ concentrations with the detection linear range of 0.1~10.0 μmol/L, giving rise to a detection limit of 24 nmol/L(3σ). Spiking recoveries ranged from 91~120% were achieved aiming at the drinking water, tap water and lake water, confirming the potential applications of the proposed method for Cu2 + detection in reality.2)The kanamycin-binding DNA aptamer(5’-TGG GGG TTG AGG CTA AGC CGA-3’) was reported firstly, which could form stable parallel G-quadruplex DNA(G4-DNA) structures by themselves and this phenomenon could be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, a novel label-free strategy for kanamycin detection was developed based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange(TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent after binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA–TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. These results could also serve as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.