Fluorescence Biosensors Based On Silver Nanoclusters And Nucleic Acid Dye To Detect Hydrogen Peroxide And Glucose

Silver nanoclusters are one kind of nanometer materials, which have special physical, chemical and optical properties. The ultra-small size of silver nanoclusters make it have many excellent properties, which are different from nanocrystals and metal atoms. These properties make up the shortages of nanocrystals and metal atoms. The excellent properties of silver nanoclusters include stable optical property, low toxicity, good biocompatibility, strong resistance to bleaching, high fluorescence quantum yields and so on. Silver nanoclusters have been widely applied in the detection of metal ions, DNA or RNA, biological small molecules, proteins, and used for cell markers and biological imaging, etc.Nucleic acid dye is a kind of dye, which is able to combine with nucleic acids and present special optical properties. At present, there are many kinds of nucleic acid dyes, such as ethidium bromide （EB）, SYBR Green I, SYBR Green II, SYBR Gold （SG）, GoldView and so on. Nucleic acid dye is widely used in biological analysis and gel electrophoresis analysis.Based on silver nanoclusters and nucleic acid dyes, we have built three new fluorescence biosensors and successfully detected hydrogen peroxide （H₂O₂） and glucose by them in this paper.（1） In chapter 2, based on the cleavage of ssDNA by ·OH which is produced by Fenton reaction and the fluorescence enhancement effect when guanine-rich （G-rich） DNA sequences are in proximity to DNA-silver nanoclusters （DNA-AgNCs）, we built a label-free fluorescence biosensor to detect H₂O₂ and glucose, and we also proved that ·OH was indeed produced by Fenton reaction in the sensing system by adding antioxidants. In the presence of ferrous iron (Fe²⁺), When H₂O₂ is introduced into the system, hydroxyl radical （·OH） is produced by the Fenton reaction. Due to the oxidative effect of ·OH, B-DNA is cleaved, leading to it cannot hybridize with G-DNA. G-DNA hybridize with Ag-DNA and the G-rich sequences are brought in proximity to AgNCs in the same time, accompanied by a significant fluorescence enhancement. H₂O₂ is sensitively detected by monitoring the change of fluorescence intensity of silver nanoclusters. Because glucose can be oxidized by O2 producing H₂O₂ in the presence of glucose oxidase （GO_x）, this fluorescence biosensor can also be used to detect glucose. This fluorescence biosensor is based on the fluorescence turn-on mode, which not only reduces the possibility of a false positive signal, but also enhances the detection sensitivity. It also have some advantages, such as without modification, low cost and so on.（2） In chapter 3, based on nucleic acid dye and the cleavage of ssDNA by ·OH which is produced by Fenton reaction, we built a label-free fluorescence biosensor to detect H₂O₂ and glucose. In the presence of Fe²⁺, when there is no H₂O₂ in the system, ssDNA interact with SYBR Gold （SG）, the fluorescence intensity enhance obviously. After adding H₂O₂ to the reaction system, Fenton reaction is taken place by H₂O₂ and Fe²⁺ and produce ·OH, due to the oxidation effect of ·OH, ssDNA is cleaved, leading to it cannot combine with SG, the fluorescence intensity of the system is not change. Therefore, H₂O₂ is successfully quantified by monitoring the change of fluorescence intensity of the system. As glucose can be oxidized producing H₂O₂ in the presence of GO_x, so this fluorescence biosensor can also quantify glucose. This sensor not only has high selectivity, simple instruments operation, low cost, but also has an ideal testing results to H₂O₂ and glucose.（3） In chapter 4, based on ssDNA conformation change which is triggered by melamine and thymine （T） forming hydrogen bonds and SYBR Green I can interact with dsDNA producing strong fluorescence, we designed a new type of label-free fluorescence biosensor for detecting H₂O₂ and glucose. In the presence of Fe²⁺, when there is no H₂O₂ in the system, stable hydrogen bonds is formed between the T of T-rich ssDNA and melamine, getting T-melamine-T specific structure, T-rich ssDNA fold into double chain structure which is similar to hair clip. SYBR Green I embed into the double chain structure, the fluorescence intensity of the system enhance obviously. After adding H₂O₂ to the reaction system, Fenton reaction is taken place by H₂O₂ and Fe²⁺ and produce ·OH, due to the oxidation effect of ·OH, ssDNA is cleaved and cannot form hydrogen bonds with melamine, SYBR Green I is free in the solution, the fluorescence intensity of the system is still very weak. By detecting the change of the fluorescence intensity in the system to detect H₂O₂. In the presence of GO_x, glucose can be oxidized producing H₂O₂, so this sensor can also be used to quantify glucose. This sensor has low cost, good selectivity and avoids complex modifications. And based on the scavenging effect of antioxidants to ·OH, we have successfully detected ascorbic acid （Vit C）.