| Composting technology is applied to disposal of the municipal solid wasteincreasingly. The metabolic process during microbial degradation of liggnocellulaseis completed together by a series of enzymes in the compost system, in whichmanganese peroxidase and cellobiohydrolase II play the key role. It is better to knowthe synergy between the two enzymes by using DNA biosensor. This study was toprepare DNA probes immobilized biosensor for detecting manganese peroxidase andcellobiohydrolase II encoding genes from Phanerochaete chrysosporium andTrichoderma reesei，respectively. The feasibility for detection of two genes wasdiscussed and polymerase chain reaction （PCR） analysis was used to study thecomposting microbial community diversity.First, the detection of manganese peroxidase encoding genes fromPhanerochaete chrysosporium was studied. Mercapto-modified probe self-assembledon the gold electrode surface and the process of immobilization and hybridization ofssDNA were characterized by impedance spectroscopy and cyclic voltammetry. Thelinear relationship of electrochemical signals and concentration of target DNA wasobtained by chronoamperometry and the range of detection, specificity, variousfactors were also studied. The amperometric current linearly related to the commonlogarithm of the target nucleic acid concentration in the range from1×10-9to1×10-15M and the detection limit of biosensor was1.0×10-17M.A magnetic separation and detection method for target sequence of a geneencoding cellulase using biocompatible core-shell nanoparticle probes was developed.Thiolated capture probe was conjugated with biocompatible Fe3O4-SiO2-Au core-shellnanoparticles. Target probe and signal probe were hybridized with capture probe onthe surface of the inorganic DNA carrier, which resulted in core-shell nanoparticleprobes. With the existence of an external magnetic field, it is convenient andtime-saving to realize the detection of cellulase gene in Trichoderma reesei （T. reesei）by liquid fermentation and the following magnetic separation. Quantitative PCR（Q-PCR）was performed to give absolute quantification to the concentration of targetnucleic acid, results of Q-PCR was compared to that of electrochemical method. Theoptimized experimental conditions were studied to maximize the hybridizationefficiency and detection sensitivity. The amperometric current response was linearly related to common logarithm of the target nucleic acid concentration in the range of1.0×10-13-1.0×10-9M, with a detection limit of1.2×10-14M.Finally, We reported a strategy for fabricating soluble multi-walled carbonnanotubes （MWCNTs） combined with carboxyl by physical and chemical methods.Anelectrodeposition method was proposed for the convalent binding of the polymerswith free amine onto MWCNTs.And then, we decorated the surface of MWCNTs/GCelectrode with nano-Au through electrochemical method. As a result,we got the genebiosensor decorated by nano-Au/MWCNTs. Due to the large specific surface area andexcellent electrochemical performance, the electrode decorated by nano-Au andMWCNTs could be better improved in the aspects of sensitivity and detection limitand thus to realize the function of testing and identifying the genes encoding lignindegrading enzymes,and also provide a basis for further discussion of enzymebiosensor applied in the detection of microbial functional genes in compost.Weinvestigated the electrochemical characteristics of the biosensor which responses tothe detected gene at the same time. The response current and the target chainconcentration within the limits of lowest1×10-11to toppest1×10-5M became a linearrelationship.It was a strong evidence that the biosensor owns a good biologicalcatalytic activity.The limit detection is1.5×10-12M. |
PDF Full Text Request