Study Of New Flourescence Assay For Protein And Small Biomolecule

There are three chapters in this thesis. Two new and simple fluorescent biosensors were established for detection of protein or small biomolecules based on RNA molecular beacon (Chapter1) and bifunctional molecular beacon (Chapter2), respectively. A new electrochemical biosensor was successfully constructed for simultaneous determination of two species in a mixture via the modification of polyaniline nanocomposite in the third chapter.1. A new strategy for determining the RNA endonuclease activity of mammalian argonaute2(Ago2) protein has been developed, which combines the unique cleavage function of Ago2protein with an RNA molecular beacon (RMB). Ago2protein and guide RNA can form RNA-induced silencing complex (RISC) which exhibites RNA endonuclesae activity. Based on the formation of RISC with RNA endonuclesae activity, the simple and sensitive detection of Ago2is achieved through the enzyme cleavage of the RMB with fluorescence restoration. The developed biosensor exhibited good selectivity due to the speciality of Ago2protein. Under the optimum conditions, the biosensor showed a well sensitivity with a linear range from1nM to25nM and a low detection limit of0.6nM.2. A bifunctional fluorescent oligonucleotide probe for small molecules and protein detection has been developed based on turn on fluorescence response via the target induced structure-switching of molecular beacon. The two loops of this molecular beacon are designed in such a manner that they consist of thrombin (Tmb) aptamer sequence and adenosine triphosphate (ATP) aptamer sequence, respectively, which are utilized to sense Tmb and ATP. The oligonucleotide forms double stem-loops in the absence of targets, yielding no fluorescence emission because of the FRET from the excited fluorophore to the proximal quencher. Upon addition of the target, the ATP or Tmb, its specific interaction with loop sequence of the hairpin structure induce the separation of reporter fluorophore and the fluorescence quencher of the molecular beacon, resulting in an increase of fluorescence response. Hence, the separate analysis of ATP and Tmb could be realized through only one designed molecular beacon. The detection limits were estimated to be25nM for ATP and12nM for Tmb, respectively. The results of this study should substantially broaden the perspective for future development of oligonucleotide probe for analysis of other analytes.3. Using three-layer SiO2@Au nanoparticles@polyaniline (SiO2@AuNPs@PANI) nanocomposite, a new electrochemical sensing platform was developed for simultaneous determination of uric acid (UA) and ascorbic acid (AA). For UA and AA in mixture, it can well be separated from the other with a potential difference of384mV, which is large enough for the selective and simultaneous determination of two species in a mixture. The DPV peak cerrents obtained in mixture increased linearly over concetrations of UA and AA in the range of5.0×10-6to1.1×10-3M and1.0×10-5to1.05×10-3M with relative coefficients of0.9989and0.9987, respectively. The detection limits for UA and AA are2×10-6and6×10-6M, respectively.