Study On The Novel Biosensor Method Based On Nano Mate Rials And Cytochrome C

The unique structure of nanomaterials endows themselves numerous special effects, including quantum size effect, small size effect and macroscopic quantum tunneling effect. As a branch of nanomaterials, glowing metal nanomaterials have attracted much attention in the area of biosensor due to their characteristics of simple synthesis, high quantum yield, excellent biocompatibility and photostability.Cytochrome c （Cyt c） is a kind of heme containing metalloprotein, which can accept electrons. The isoelectric point （PI） of Cyt c is around 10.0, so it is positively charged when the pH of buffer is lower than its PI. Armed with these two attractive features, Cyt c has been widely used in the field of biosensor.In this thesis, based on Cyt c and nanomaterials （silver nanoclusters and copper nanoparticles）, we have developed three novel fluorescent biosensor platforms. The followings are the details:（1） In chapter 2, we proposed a new method for trypsin detection based on the electron transfer between Ag NCs and Cyt c. Ag NCs are synthesized by one-pot method using oligonucleotide as the template, and they are negatively charged because of the abundant phosphate groups. The PI of unbroken Cyt c is about 10.0, so it’s positively charged under this experimental condition. When Ag NCs and Cyt c exist simultaneously, they form a complexe via electrostatic interactions, then the fluorescence of Ag NCs is quenched by heme in the Cyt c. Trypsin can hydrolyze Cyt c to small peptide fragments. The PI of heme-associated peptide fragment is around 7.0, and it is slight negatively charged, thus has little impact on Ag NCs, as a result Ag NCs keep a strong fluorescence in solution. The detection of trypsin can be realized by measuring the fluorescence intensity. In addition, trypsin inhibitors have the ability to inhibit the activity of trypsin, so the sensor can be further used for trypsin inhibitors screening. This method has the advantages of simple synthesis, no label, low cost as well as good selectivity.（2） In chapter 3, we developed a novel DNA biosensor based on Cyt c and Exo Ⅲ-assisted enzymatic recycling amplification strategy. The main point of this sensor is that Cyt c has different quench abilities towards DNA-associated fluorophore and single fluorophore due to different affinities. A ssDNA, which modifies with a fluorophore at its 5’-terminus is chosen as a fluorescent probe. In the absence of target DNA, the probe can’t act as substrate for Exo Ⅲ cleavage. The undigested signaling probe will bind to Cyt c by electrostatic interactions. Then, the fluorescence of the fluorophore is quenched by the heme in Cyt c. In the presence of target DNA, the probe hybridizes with target and forms a duplex, which is able to be hydrolyzed into mononucleotides by Exo Ⅲ, liberating fluorophore and target. The released target hybridizes with another probe, and the cycle starts anew, while the released fluorophore has a weaker affinity with Cyt c, so it keeps in solution to show a fluorescence signal. This method is capable to differentiate single-base mismatch. Meanwhile, simultaneous detection of two different targets can be achieved.（3） In chapter 4, we developed a platform for melamine detection by taking advantage of the property that melamine can form hydrogen bonds with thymine, and then inhibit the synthesis of fluorescent copper nanoparticles （Cu NPs）. The selected nucleic acid sequence T30 is comprised of 30 thymines. The premise of formation of T30-temp led Cu NPs is that Cu²⁺ is able to adsorb on DNA, then Cu²⁺ is reduced to Cu0 by sodium ascorbic acid along the contour of DNA structural framework. After melamine form hydrogen bonds with thymine, the two processes mentioned above are both hampered. And this interruptive effect increases upon increasing the melamine concentration within a certain range, as a result the fluorescence intensity of the as-prepared Cu NPs decreases. By monitoring the fluorescence signal, quantitative detection for melamine can be achieved. This method is rapid, reliable and simple.