Study On The Chemiluminescence Catalytic Activity Of Cationic Liposomes And Its Applications

CL analysis has received increasing attention in different areas including clinical diagnosis,food safety,and environmental assessment due to its characteristics of high sensitivity,wide linear range,fast analysis speed,and low price.Luminol-H₂O₂ CL reaction is the most commonly used CL system.However,the CL intensity of this CL reaction is weak in the absence of a catalyst.Several kinds of catalysts for the luminol-H₂O₂ CL reaction have been developed,including peroxidases,metal ions,and nanomaterial.Peroxidases and metallic ions such as Cr³⁺,Co²⁺,Au³⁺,etc.are classical catalysts for the luminol-H₂O₂ CL reaction over the past few years.Nowadays,more and more nanomaterial as mimetic peroxidases were introduced in the luminol CL reaction.Liposomes are spherical phospholipid bilayer vesicles that are commonly used in drug delivery systems.In the analytic field,various enzymes were encapsulated in the aqueous compartment of liposomes which is surrounded by lipid bilayer membranes.Various enzyme-encapsulated liposomes were developed and employed as biocompatible catalysts,biosensing materials,immunoassay labels,etc.Although liposomes were widely used previously,to the best of our knowledge,the investigation of liposomes especially positive charged liposomes as catalysts for the luminol CL reaction has not been reported yet.We found an interesting phenomenon in the experiment.The positive-charged DOTAP liposomes exhibited a good catalytic activity on the luminol-H₂O₂ CL reaction while the negative-charged liposomes and neutral liposomes showed a negligible effect on the luminol CL reaction.Therefore,in this thesis,the DOTAP cationic liposomes-catalyzed luminol-H₂O₂ CL system was established and it was applied for the detection of specific sequences of DNA and adenosine triphosphate（ATP）.The details of the study were as follows.In chapter 2,we established the DOTAP cationic liposomes-catalyzed luminol-H₂O₂ CL reaction and investigated the CL mechanism.Mechanism studies showed that the positive charge on the surface of liposomes plays an important role in the CL process.Cationic liposomes may be capable of catalyzing the decomposition of H₂O₂ leading to the production of^·OH,then the^·OH radicals reacted with HO₂^-to facilitate the formation of O₂^·-.The produced O₂^·-may further react with^·OH radicals leading to the formation of ¹O₂.These oxygen-related radical species may be surrounded by the surface of cationic liposomes.Because of the electrostatic interaction between the cationic liposomes and luminol anion,the luminol anions tended to move close to the surface of the cationic liposomes and then to be oxidized by the oxidizing radical species which may be around the surface of cationic liposomes forming 3-aminophthalate*（3-APA*）.When the excited-state 3-APA returned to the ground-state,an enhanced CL was observed.We also optimized the experimental conditions for the DOTAP cationic liposomes-catalyzed luminol-H₂O₂ CL reaction and obtained the standard curve of cationic liposomes.Under the optimized experimental conditions,the linear relationship between the CL intensity and the logarithmic concentration of cationic liposomes ranging from 2.83 to 362.5μg m L^-1was obtained.The effects of some metal ions and 20 kinds of amino acids on the luminol-H₂O₂-cationic liposome CL system were investigated.Among them,three kinds of amino acids inhibited the CL intensity.Glu and Asp are negatively charged in water,which decrease the positive charges on the cationic liposomes,thus,resulting in the inhibition of CL signals.The inhibition effect of Cys was attributed to its reducibility.The proposed CL reaction may be applied to the detection of these amino acids.In chapter 3,we investigated the effect of negative-charged single-strand DNA on the luminol-H₂O₂-cationic liposome CL system and established a label-free CL detection platform for the detection of sequence-specific DNA.We found that negative-charged single-strand DNA exhibited a good inhibition effect on the luminol-H₂O₂-cationic liposomes CL reaction.The CL intensity decreased linearly with an increasing amount of DNA from 0.05 to 2 pmol.We optimized the amount of capture DNA for the detection of sequence-specific DNA.Under the optimal conditions,the linear relationship between the CL intensity and the amount of target DNA from 0.5 to 8 pmol was obtained.The label-free CL platform can differentiate the perfect matched target DNA from the two-base mismatched oligonucleotides.Compared to the full matched target DNA,the single-base mismatched DNA showed a 51.2%hybridization efficiency.Further improvement of the selectivity may be achieved by stringent control of temperature during washing steps.In chapter 4,base on the ss DNA-inhibited luminol CL system,we established a label-free CL platform for the detection of ATP as a model analyte using anti-ATP aptamer as a recognition element.We optimized the aptamer amount and the incubation temperature between ATP and ATP aptamer.Under the optimal experimental conditions,a linear relationship between CL intensity and ATP amount in the range of 1.25-40 nmol was obtained.The proposed label-free ATP CL assay method can be applied to the detection of ATP in serum samples.The recovery of 20 nmol and 40 nmol ATP ranged from 90.1%-132.3%.