Novel Fluorescent Biosensing Method Based On Nucleic Acid Dye And Copper Nanomaterials

Fluorescence technique with the use of functional dyes and biomarkers makes tests of many basic processes in life science be realized with high efficiency, high sensitivity, reliability and reproducibility, such as the interaction between molecules or ions in life activities, etc. Sensitively detection of trace amounts of target analytes in biological samples usually need to mark probe with fluorescence groups or embed fluorescent dyes into it, and SYBR Green I is an example. Making use of the properties that SYBR Green I which is a kind of new functional dyes can combine with nucleic acid and emit fluorescence, we can detect trace materials by the combination of SYBR Green I with a probe.In addition, nanomaterials with unique quantum size effect and surface structure effect have a series of superior properties, so that many ordinary materials couldn't compare with them. And this thesis makes use of the properties of metal nanomaterials, a branch of nanomaterials, as fluorescent markers to analyse small molecular substances. Metal nanomaterials have many excellent properties, such as easy synthesis, good biocompatibilities, which are of great scientific interest, and copper nanoparticles are one of them. It has the characteristics of simple synthesis, short preparation time, different fluorescence intensity with adjustable particle sizes, making it suitable for the improvement of fluorescence biosensor and sensing method, and received more attention in the study of the application of biosensor.To sum up, in order to make improvements to the stability and sensitivity of biosensors, this paper constructed several groups of fluorescent biosensors for the study of adenosine triphosphate, trypsin and alkaline phosphatase. The main contents are as follows:(1) In Chapter 2, a G-quadruplex-based, label-free fluorescence assay was demonstrated for the detection of adenosine triphosphate (ATP). First, a double-stranded DNA (dsDNA), hybridized by ATP-aptamer and its complementary sequence, was a nucleic acid for the combination of ATP. Besides, SYBR Green ? (SG ?) nucleic acid dye is used as a fluorescent probe of this method, at the same time, the addition of exonuclease ? (Exo ?) reduces the background signal of this scheme. In the absence of ATP, SG I will embed into double-stranded DNA which was then digested by Exo ?, resulting in a low background signal. When in the presence of ATP, the ATP aptamer in double-stranded DNA meets with ATP molecules will fold into G-quadruplex structure which contains ATP molecules in it, and the formation of the G-quadruplex structure resisted the hydrolysis of Exo ?. SG I was then inserted into the above structure, showing significantly enhanced fluorescence signal compared to which in aqueous solution. Owing to a decrease of the background noise, a high signal-to-noise ratio could be obtained for this scheme. This sensor can detect ATP with a concentration ranging from 50 ?M to 5 mM, and with a detection limit of 5 ?M.(2) In Chapter 3, we use copper nanoparticles (CuNPs) as a fluorescence probe, and we constructed a fluorescent biosensing strategy for the detection of trypsin with a low concentration of cytochrome c (Cyt c) as the hydrolysis substrate of trypsin. Whereas, here a low concentration of Cyt c was designed especially for that it avoids the fluorescence quenching of CuNPs caused by the electron transfer process from Cyt c to CuNPs. In the presence of trypsin, Cyt c would be hydrolyzed to small peptides, releasing free cysteine residues. Nonfluorescent coordination complexes were formed upon exposure to free cysteine residues by metal-ligand bond between Cu atoms and sulfur atoms, leading to a decreased fluorescence response to CuNPs. This novel method for quantitative determination of trypsin has a linear detection range from 0.25 ?g ml/1 to 1000 ?g mL-1 and a relatively low detection limit of 42 ng mL-1.(3) In Chapter 4, an approach for the detection of alkaline phosphatase (ALP) was developed without the need of labeling. This approach relies on the inhibition of poly (thymine)-templated fluorescent copper nanoparticles (CuNPs) by pyrophosphate (PPi), which was a turn-on fluorescent method. This approach depends on the strong affinity of PPi to Cu2+, which would hamper the effective formation of fluorescent CuNPs, leading to a low fluorescence intensity of the system. ALP was used as the hydrolytic enzymes of PPi, which would disable the complexation between Cu2+ and PPi, facilitating the formation of fluorescent CuNPs. As a result, the fluorescence enhancement of the system was positively related to the concentration of ALP. This method is cost-effective and convenient without any labels or complicated operation process. The present strategy exhibits a high sensitivity and a high selectivity for the sensing of ALP. Additionally, we also studied the inhibition effect of phosphate to the hydrolysis of ALP. The proposed method using a PPi substrate may have some potential application values in the diagnosis of diseases associated with ALP enzymes.