Investigation Of New Fluorescent Biosensors Based On Copper Nanoparticles And Molecular Beacon

As a new analytical technique involving many subjects, biosensing technique hasbeen developing rapidly in recent years. Due to many advantages such as goodselectivity, high sensitivity, fast response, low cost and continuous on-line detection incomplex system, biosensors have been widely used in medical diagnostics, foodsecurity, terrorism, and so on. With the continuous development of the analyticalscience, the ability of qualitative and quantitative analysis for various biomolecules indifferent conditions is increasingly required. The discovery of functional nucleic acidsand nanomaterials has provided an interesting alternative to biosensing system, andresulted in a breakthrough in such traditional sense, including sensitivity, selectivity,etc. Based on the above considerations, in this master thesis, several bioassay systemshave been developed focused on new methods for enhancing the performance ofbiosensors by using double-stranded DNA-copper nanoparticles and molecular beacon(MB). The details are summarized as follows:(1) Study on double stranded-copper nanoparticles based biosensor. Coppernanoparticles exhibit fascinating features including ease of synthesis, good wate rsolubility, low toxicity, and biocompatibility. Double-stranded DNA-coppernanoparticles based sensors always exhibit excellent performance. Thus, in chapter2,using double-stranded DNA-copper nanoparticles as signal reporters, a label-freebiosensor for the sensitive detection of nuclease was developed. The double-strandedDNA could act as an efficient template for the formation of copper nanoparticles withhigh fluorescence, whereas the single-stranded DNA cannot support the formation ofcopper nanoparticles. This difference in fluorescent signal generation can be used forthe detection of nuclease activity. The enzyme S1nuclease was taken as the modelanalyte. S1nuclease is known to be a single-strand-specific endonuclease. Upon theaddition of the S1nuclease into the sensor, the DNA was cleaved into fragments,preventing the formation of the copper nanoparticles and resulting in low fluorescence.Under the optimized experimental conditions, the sensor exhibit ed excellentperformance (e.g., a detection limit of0.3U mL1). In chapter3, a new and facilebiosensor using double-stranded DNA-copper nanoparticles as fluorescence reportersfor the highly sensitive and selective detection of L-Histidine was demonstrated. In thedouble-stranded DNA-copper nanoparticles sensor the fluorescence could be quenched considerably upon the addition of L-Histidine. The luminescence quenching in thepresence of L-histidine may be attributed to the complexation between L-histidine andthe Cu2+ion, since the concentration of Cu2+ion was an important parameterinfluencing the fluorescent probes of double-stranded DNA-copper nanoparticles,therefore, L-histidine could influence the fluorescence of double-stranded DNA-coppernanoparticles. Under the optimized experimental conditions, the sensor exhibitedexcellent performance (e.g., a satisfactory detection limit of5μM). Moreover, theproposed biosensor could be applicable for the detection of target biomolecule incomplex biological samples.(2) Study on molecular beacon based biosensor. In chapter4, A universalbiosensor for fluorescence turn-on detection of biomolecules was developed based onFenton reaction triggered molecular beacon cleavage. In the presence of glucose, theglucose oxidase would bind with it and catalyze the oxidation reaction generating H2O2.The generated H2O2was further decomposed to produce OH through theFe2+-catalyzed Fenton reaction. Then, in-situ-generated OH could trigger the cleavageof the MB, and the fluorescence intensity would be dramatically increased because ofthe complete separation of the fluorophore from the quencher. By employing molecularbeacon as both recognition and reporter probes to low background signal, the proposedsensor showed high sensitivity to targets. It also exhibited high selectivity，which madeit valuable for the detection of target biomolecule in complex biological samples. Toverify the universality of the sensing system based on molecular beacon cleavage, thestrategy was further applied to the detection of choline with ChOx instead of GOx asthe oxidase. A detection limit of1μM was estimated for choline.