Studies On Methods Of Peptidase Detection And Fluorescent Labelling Of Target Cell Surfaces

Biosensor as a kind of modern sensing technology is an interdisciplinary andintegrated research including physical, chemistry, biology, elect ronics, information andcomputing science. With biological sensing technology facilitating the remarkable andeven revolutionary development of life science, it has drawn more and more attentionfrom many analytical chemist, as the most potential and practical new biologicalsensing technology in the21st century. Compared with conventional assay, by thevirtue of high specificity, good sensitivity, small volume, low cost, real-time andcontinuous on-line analysis, biosensors have a wide application in biochemicalanalysis, medical examination, food security, health care, agriculture, animalhusbandry, environment monitoring, military and other relevant fields.The thesis focuses on the activity detection of peptidase and fluorescent labellingof target cell surfaces. Immunochemistry principle and specific recognition of aptamerare used together with enzyme catalysis and hybridization chain reaction signalamplification to build versatile biosensor in a variety of signal transduction ways. Inorder to improve the performance and expand the application range of biosensors, thedetails of this dissertation are described as follows:(1) In chapter2, a novel platform for detection of histone deacetylase (HDAC)activity has been developed based on gold nanoparticles (Au NPs) based fluorescenceresonance energy transfer (FRET) immunoassay. This strategy combines the acetylatedfluorescent peptide probe with the anti-acetyl antibody functionalized Au NPs tomeasure the deacetylation activity of histone deacetylase sirtuin2(SIRT2). Enzymaticdeacetylation of the acetylated peptide substrate is detected by means of Au NPslabeled anti-acetyl peptide antibody with the formation of the immunocomplexresulting in energy transfer between the fluorescent dyes and the Au NPs. Due to thehighly efficient fluorescence quenching of Au NPs, the proposed method shows a lowbackground and favorable sensitivity and the fluorescence peak intensity is linearlydependent on the SIRT2concentration in a range from50-500nM. The detection limitwas estimated to be11.9nM, according to the3rule. In addition, this approach canbe applied to the evaluation of HDAC inhibitor activity. The proposed platform shouldpotentially facilitate the development of new assays for HDAC activity and otherhistone modifications. (2) In chapter3, a sensitive and convenient electrochemical immunobiosensor forthe detection of histone deacetylase based on acetylated peptide with protein enzymecatalysis has been constructed. This strategy combines immunochmistry technoloywith catalyzing amplification to measure the deacetylation activity of histonedeacetylase sirtuin2(SIRT2). Enzymatic deacetylation of the acetylated peptidesubstrate is detected by means of rabbit anti-acetylated peptide antibody as firstantibody with the formation of the immunocomplex, followed by alkaline phosphateconjugated goat anti-rabbit antibody (ALP-secondary antibody) through specificcombination interaction to produce amplified signal electrochemical. SIRT2is chosenas target model, and due to deacetylation of SIRT2, the proposed immunosensor showsa decreased electrochemical signal. The proposed strategy exhibits a good analyticalperformance and possesses such advantages as good sensitivity, simplicity andconvenience. The electrochemical signal response is linearly dependent on the SIRT2concentration in a wide range from1-500nM. The detection limit was estimated to be0.1nM, according to the3rule. In addition, this approach can also be applied to theevaluation of HDAC inhibitor activity and inhibitor screening. The proposed platformholds great potential for the development of new methods for HDAC activity andhistone modifications.(3) In chapter4, chemically-modified and label-free fluorescent DNA nanosensorhave been built for anchoring on target living cell surfaces. The cell membrane isinvolved in many cellular processes such as cell adhesion/signaling, and it se rves as anattachment site for numerous extracellular structures. Thus, the ability to modify cellmembrane has become essential for the comprehensive understanding and adequateregulation of the biological properties. The ability to build analytic or regu latory toolsable to deftly navigate this complex microenvironment is critical but not easy. Thischallenge motivated us to design nanoscale devices that can be attached to orself-assembled on target living cell surfaces. Here we report the anchoring ofpreformed chemically-modified and label-free fluorescent DNA nanodevices on targetliving cell surfaces. To achieve the goal, the fluorescent DNA nanosensors was builtusing an aptamer-tethered DNA nanotrain (aptNTr) platform. These aptNTrs wereself-assembled through a hybridization chain reaction (HCR) and anchored on cellsurfaces by target-specific aptamer recognition. As a tool for bioanalysis andbioregulation, these fluorescent DNA nanosensors were capable of exhibitingfluorescence emission and undergoing imaging on target living cell surfaces forpinpoint analysis and, importantly, regulation of biological activities.