The Construction And Application Research Of Polypeptide-based Fluorescent Biosensor

In this thesis, under the research background of the polypeptidefunctionalized nanomaterials-based fluorescence biosensor, the peptidechains with specific sequence were designed and synthesized, and theinteraction mechanism between the polypeptides and the nanomaterialswas explored, which had been successfully applied for the detection ofprotease activity. The contents are as follows:First of all, the characteristics and research progress of fluorescentbiosensor and polypeptides were reviewed. The reported interactionmechanism between polypeptides and biological molecules wassummarized and the application progress of the polypeptides in proteasefluorescent sensor was analyzed. The contents of this paper wereexpounded and the working assumption was presented.Secondly, the water-soluble CdTe quantum dots (QDs) usingmercaptopropionic acid as modifier were prepared and their spectroscopicproperties and morphology were characterized. A novel and uniquepeptide chain was designed for the first time, and it can combine with thewater-soluble semiconductor nanocrystals to induce their aggregation andalso can be hydrolyzed by trypsin. Therefore, a simple, label-free andsensitive turn-on fluorescence sensor for trypsin was build with adetection limit of20ng/mL, and the naked eye detection could be realizedunder the UV light. Thirdly, through summarizing the above works, we found that thesimple multiplexing and high-throughput method for determination ofprotease activity has more practical application value for rapid medicaldiagnostic. Thus we took advantages of the aggregation behavior of theQDs induced by FAM labeled peptide to construct a FAM-peptide-QDsbased multiplexed fluorescence sensor for monitoring the activity oftrypsin and collagenase. In the as-constructed biosensor, a single peptidesubstrate was used for examining the two enzymes, further expanding theapplication efficiency of multiplexed protease sensor. The method issimple, the linear range for trypsin and collagenase detection was from0.75μg/mL to15μg/mL and from5μg/mL to50μg/mL, respectively andthe detection limit was0.2μg/mL and1μg/mL, respectively.At last, a new high-sensitive collagenase fluorescent biologicalsensor was developed using of graphene oxide (GO) because of its largespecific surface area, good water solubility and biocompatibility. Thelinear range of the method for collagenase detection was from0.5μg/mLto40μg/mL, nearly two orders of magnitude, and the detection limit was0.2μg/mL.