Analysis Of Biological And Chemical Poisonous Substances Using Functionalized Nanomaterials

In this thesis,we developed several novel methods for the detection of biological and chemical poisonous substances such as Magnaporthe oryzae,copper ions,exonuclease III activity and cancer cells etc based on the functionalized nano-materials.The main contents of this thesis included:(1)We have expressed and purified the M.oryzae's chitinases(Mgchi)and a rice cDNA encoding mannose-binding jacalin-related lectin(Osmbl)with.related genes.We demonstrated that Mgchi could be used as a biochemical marker for the detection of M.oryzae and there was a specific interaction between Osmbl and Mgchi.Using Mgchi as biochemical marker and Osmbl as recognition probe,we developed a visual method for the specific and sensitive detection of M.oryzae based on the PdNPs-catalyzed TMB/H2O2 system.The proposed method could be used to detect as low as 7.5×10-9 M of Mgchi by naked eye observation and 2.5×10-11 M of Mgchi by the micro-plate reader.With the help of the method,we had successfully detected M.oryzae in the real M.oyzae-infected rice plant with recoveries of 88-109%and RSD<7%.The proposed method was sensitive,specific,potentially high throughput and cost-effective.The success in this study provides a promising substitution for the early diagnosis and fast screening of M.grisea in rice plant.(2)A novel magnetic-controllable electrochemical biosensor for the ultra sensitive and specific detection of M.oryzae in rice plant by using M.oryzae's chitinases(Mgchi)as biochemical marker and a rice(Oryza sativa)cDNA encoding mannose-binding jacalin-related lectin(Osmbl)as recognition probe was reported.The proposed biosensor combined with the merits of chronoamperometry,electrically magnetic controllable gold electrode and magnetic beads(MBs)-based palladium nano-particles(pdNPs)catalysis amplification,has an ultra-high sensitivity and specificity for the detection of trace M.oryzae in rice plant.The detection limit of the method is 17 pg/mL(0.42 pM).It could be used to detect M.otyzae in rice plant in the initial infection stage(before any symptomatic lesions were observed)to help farmers timely manage the disease.In comparison with previous methods,the proposed method has notable advantages such as higher sensitivity,excellent specificity,short analysis time,robust resistibility to complex matrix and low cost etc.The success in this study provides a reliable approach for the early diagnosis and fast screening of M.oryzae in rice plant.(3)A novel phosphatidylserine-functionalized AuNPs(gold nanoparticles)was reported for the visual detection of Cu2+ by employing phosphatidylserine for Cu2+recognition and AuNPs for signal generation.The phosphatidylserine(1,2-dioleoyl-sn-glycero-3-phospho-L-serine,DOPS)was covalently assembled on the AuNP surface to obtain DOPS-functionalized AuNPs.It was demonstrated that DOPS-functionalized AuNPs could specifically bind Cu2+ and lead to the aggregation of AuNPs,which gave rise to a colour change from wine-red to blue and a new absorption band around 650 nm.This provides a sensing platform for the simple,rapid and field portable colorimetric detection of Cu2+.By using the sensing platform,a selective and sensitive visual biosensor for the detection of Cu2+ was developed.The proposed biosensor has outstanding analytical advantages such as good stability,relatively high sensitivity,low cost and short analysis time.It can be used to detect as low as 30?M of Cu2+ in river water by observation with the naked eye and 1.55?M of Cu2+ in river water by UV-visible spectrophotometry,within 10 min and with a recovery of 98-103%and a relative standard deviation(RSD)<4%(n=6).The proposed biosensor is promising for on-site detection of trace Cu2+ in clinical diagnosis or environmental monitoring.(4)A "turn-on" and label-free fluorescent assay for the specific,rapid,and sensitive detection of 3'5'?exonuclease III activity was reported in this study.The assay was based on the Tb3+-promoted G-quadruplex,which lead to the enhancement of Tb3+ fluorescence due to the energy transfer from guanines.The proposed assay is highly simple,rapid,and cost-effective,and does not require sophisticated experimental techniques such as gel-based equipment or radioactive labels.It can be used for the rapid detection of exonuclease III activity with a detection limit of 0.8 U and a relative standard deviation(RSD)<5%(n=6).Notably,no dye was covalently conjugated to the DNA strands,which offers the advantages of low-cost and being interference-free.(5)A novel method for sensitive and specific counting cancer cells by using MBs-based AuNPs-aptamer labeling technique and ICP-MS detection was reported.The MBs-based AuNPs-aptamer labeling technique can specifically recognize cancer cell and tag AuNPs on the cancer cells,which provides a platform for indirectly counting cancer cells via detecting Au with ICP-MS.The method reported in this study combined the merits of MBs-based AuNPs-aptamer labeling technique and ICP-MS detection,has excellent specificity,high sensitivity,excellent stability and short analysis time.Especially,the employment of ICP-MS detection and the presence of MBs,which facilitates the magnetic separation of target cells,not only greatly shorten analysis time but also greatly enhance the resistance of our method to the complicated matrix.The proposed method can be used to count as low as 100 cells/mL of human hepatocellular carcinoma SMMC-7721 cells in the serum with a recovery of 93-103%and a relative standard deviation(RSD)<4%(n=5).The success of this study provides a promising approach for the early point-of-care detection of human hepatocellular carcinoma cell in clinical diagnosis.